A Preview of the Cell

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CELS191Cell and Molecular Biology CHAPTER 1 A Preview of the CellA Publication from Pearson Custom Publishing exclusively for the University of Otago page 17

The Cell Theory: A Brief History 1

The cell is the basic unit of biology. Every organism ei-ther consists of cells or is itself a single cell. Therefore, it isonly as we understand the structure and function of cellsthat we can appreciate both the capabilities and thelimitations of living organisms, whether animal, plant, ormicroorganism.

We are in the midst of a revolution in biology that hasbrought with it tremendous advances in our understand-ing of how cells are constructed and how they carry out allthe intricate functions necessary for life. Particularly sig-nificant is the dynamic nature of the cell, as evidenced byits capacity to grow, reproduce, and become specializedand by its ability to respond to stimuli and to adapt tochanges in its environment.

Cell biology itself is changing, as scientists from a va-riety of related disciplines focus their efforts on the com-mon objective of understanding more adequately howcells work. The convergence of cytology,genetics,and bio-chemistry has made modern cell biology one of the mostexciting and dynamic disciplines in contemporary biol-ogy.

In this chapter, we will look briefly at the beginningsof cell biology as a discipline. Then we will consider thethree main historical strands that have given rise to ourcurrent understanding of what cells are and how theyfunction.

The Cell Theory: A Brief History

The story of cell biology started to unfold more than 300years ago, as European scientists began to focus theircrude microscopes on a variety of biological materialranging from tree bark to human sperm. One such scien-

tist was Robert Hooke, Curator of Instruments for theRoyal Society of London. In 1665, Hooke used a micro-scope that he had built himself to examine thin slices ofcork cut with a penknife.He saw a network of tiny boxlikecompartments that reminded him of a honeycomb.Hooke called these little compartments cellulae, a Latinterm meaning little rooms. It is from this word that weget our present-day term, cell.

Actually, what Hooke observed were not cells at allbut the empty cell walls of dead plant tissue, which is whattree bark really is. However, Hooke would not havethought of his cellulae as dead, because he did not under-stand that they could be alive! Although he noticed thatcells in other plant tissues were filled with what he calledjuices, he preferred to concentrate on the more promi-nent cell walls that he had first encountered.

One of the limitations inherent in Hookes observa-tions was that his microscope could only magnify objects30-fold, making it difficult to learn much about the inter-nal organization of cells. This obstacle was overcome a few

years later by Antonie van Leeuwenhoek, a Dutch shop-keeper who devoted much of his spare time to the designof microscopes. Van Leeuwenhoek produced hand-polished lenses that could magnify objects almost 300-fold. Using these superior lenses, he became the first toobserve living cells, including blood cells, sperm cells, andsingle-celled organisms found in pond water. He reportedhis observations to the Royal Society in a series of papersduring the last quarter of the seventeenth century. His de-tailed reports attest to both the high quality of his lensesand his keen powers of observation.

Two factors restricted further understanding of thenature of cells. One was the limited resolution of themicroscopes of the day, which even van Leeuwenhoeks

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CHAPTER 1 A Preview of the Cell CELS191Cell and Molecular Biologypage 18 A Publication from Pearson Custom Publishing exclusively for the University of Otago

2 Chapter 1 A Preview of the Cell

The challenge of understanding cellular structure and organiza-tion is complicated by the problem of size. Most cells and their

organelles are so small that they cannot be seen by the unaidedeye. In addition, the units used to measure them are unfamiliarto many students and therefore often difficult to appreciate. Theproblem can be approached in two ways: by realizing that there

are really only two units necessary to express the dimensions ofmost structures of interest to us, and by illustrating a variety of

structures that can be appropriately measured with each ofthese units.

The micrometer is the most useful unit for expressingthe size of cells and larger organelles.A micrometer (sometimes

(mm)

Units of Measurement in Cell Biology

B o x 1 A

10 m

ChloroplastPlant cell

(20 30 m)

Animal cell

(20 m)

Bacterium

(1 2 m)

Mitochondria

Nuclei

Vacuole

Figure 1A-1 The World of the Micrometer. Structures with dimensions that can be measuredconveniently in micrometers include almost all cells and some of the larger organelles, such asthe nucleus, mitochondria, and chloroplasts.

superior instruments could push just so far. The secondand probably more fundamental factor was the essentiallydescriptive nature of seventeenth-century biology. It wasbasically an age of observation, with little thought given toexplaining the intriguing architectural details of biologicalmaterials that were beginning to yield to the probing lensof the microscope.

More than a century passed before the combinationof improved microscopes and more experimentallyminded microscopists resulted in a series of developmentsthat culminated in an understanding of the importance of

cells in biological organization. By the 1830s, improvedlenses led to higher magnification and better resolution,such that structures only 1 micrometer apart couldbe resolved. (A micrometeris or one-millionth ofa meter; see Box 1A for a discussion of the units of mea-surement appropriate to cell biology.)

Aided by such improved lenses, the English botanistRobert Brown found that every plant cell he looked at con-tained a rounded structure, which he called a nucleus, a

10:6 m,(mm)

term derived from the Latin word for kernel. In 1838, hisGerman colleague Matthias Schleiden came to the impor-tant conclusion that all plant tissues are composed of cellsand that an embryonic plant always arises from a singlecell. Similar conclusions concerning animal tissue were re-ported only a year later by Theodor Schwann, thereby lay-ing to rest earlier speculations that plants and animalsmight not resemble each other structurally. It is easy to un-derstand how such speculations could have arisen. Afterall, plant cell walls provide conspicuous boundaries be-tween cells that are readily visible even with a crude

microscope, whereas individual animal cells, which lackcell walls, are much harder to distinguish in a tissue sam-ple. It was only when Schwann examined animal cartilagecells that he became convinced of the fundamental similar-ity between plant and animal tissue, because cartilage cells,unlike most other animal cells, have boundaries that arewell defined by thick deposits of collagen fibers. Schwanndrew all these observations together into a single unifiedtheory of cellular organization, which has stood the test of


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The Emergence of Modern Cell Biology 3

also called a micron) corresponds to one-millionth of a meterIn general, bacterial cells are a few micrometers in

diameter, and the cells of plants and animals are 10- to 20-foldlarger in any single dimension. Organelles such as mitochondriaand chloroplasts tend to have diameters or lengths of a fewmicrometers and are therefore comparable in size to whole bac-terial cells. Smaller organelles are usually in the range of

As a rule of thumb, if you can see it with a lightmicroscope,you can probably express its dimensions conve-niently in micrometers, since the resolution limit of the lightmicroscope is about Figure 1A-1 illustrates avariety of structures that are usually measured in micrometers.

The nanometer (nm), on the other hand, is the unit ofchoice for molecules and subcellular structures that are too

small or too thin to be seen with the light microscope.Ananometer is one-billionth of a meter It takes 1000nanometers to equal 1 micrometer. (An alternative to the termnanometer is therefore millimicron, ) As a benchmark onthe nanometer scale, a ribosome has a diameter of about2530 nm. Other structures that can be measured convenientlyin nanometers are microtubules, microfilaments, membranes,and DNA molecules. The dimensions of these structures areindicated in Figure 1A-2.

Another unit frequently used in cell biology is the angstrom() which corresponds to or 0.1 nm. Molecular dimen-sions, in particular, are often expressed in angstroms. However,

because the angstrom differs from the nanometer by only a fac-tor of ten, it adds little flexibility to the expression of dimen-sions at the cellular level and will therefore not be used in thistext.

10:10 m

mm.

(10:9 m).

0.200.35mm.

0.21.0 mm.

(10:6 m).

78 nm

25 nm

Bacterial ribosome

25 nm

Microtubule

2 nm

DNA helix

7 nm

Typical membrane

Large subunit

Small subunit

Microfilament

Figure 1A-2 The World of the Nanometer. Structures with dimen-sions that can be measured conveniently in nanometers includeribosomes, membranes, microtubules, microfilaments,and theDNA double helix.

time and continues to provide the basis for our own un-derstanding of the importance of cells and cell biology.

As originally postulated by Schwann in 1839, the celltheoryhad two basic tenets:

1. All organisms consist of one or more cells.2. The cell is the basic unit of structure for all organisms.

Less than 20 years later, a third tenet was added. This grewout of Browns original description of nuclei, extended byKarl Ngeli to include observations on the nature of celldivision. By 1855, Rudolf Virchow, a German physiologist,

concluded that cells arose in only one mannerby thedivision of other, preexisting cells. Virchow encapsulatedthis conclusion in the now-famous Latin phrase omniscellula e cellula, which in translation becomes the thirdtenet of the modern cell theory:

3. All cells arise only from preexisting cells.

Thus, the cell is not only the basic unit of structure for allorganisms but also the basic unit of reproduction. In

other words, all of life has a cellular basis. No wonder,then, that an understanding of cells and their properties isso fundamental to a proper appreciation of all otheraspects of biology.

The Emergence of ModernCell Biology

Modern cell biology involves the weaving together of threedistinctly different strands into a single cord. As the time-

line of Figure 1-1 illustrates, each of the strands had itsown historical origins, and most of the intertwining hasoccurred only within the last 75 years. Each strand shouldbe appreciated in its own right, because each makes itsown unique and significant contribution. Contemporarycell biologists must be adequately informed about all threestrands, regardless of their own immediate interests.

The first of these historical strands is cytology, whichis concerned primarily with cellular structure. (The Greek


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Allen and Inou perfect video-enhanced

contrast light microscopy

Berg, Boyer, and Cohen develop

DNA cloning techniques

Palade, Sjstrand, and Porter develop

techniques for electron microscopy

Buchner and Buchner

demonstrate fermentation

with cell extracts

Pasteur links

living organisms to

specific processes

Wohler synthesizes

urea in the laboratory

DNA sequencing methods developed

Avery, MacLeod, and McCarty show DNA

to be the agent of genetic transformation

Krebs elucidates the TCA cycle

Svedberg develops the ultracentrifuge

Embden and Meyerhof describe

the glycolytic pathway

BIOCHEMISTRY

Invention of

the microtome

Golgi

complex

described

Feulgendevelopsstain for DNA

Sutton formulatesChromosomaltheory of heredity

Roux and Weissman:Chromosomes carrygenetic information

Flemming identifieschromosomes

Van Leeuwenhoek improves lenses

Hooke describes cellulae

2000

1975

1950

1925

1900

1875

1850

1825

1800

1700

1600

First transgenic animals produced

Heuser, Reese, and colleagues develop

deep-etching technique

Genetic code elucidated

Kornberg discovers DNA polymerase

Watson and Crick propose double helix for DNA

Hershey and Chase establish DNA as the genetic material

Claude isolates first mitochondrial fractions

Invention of the electron microscope

Levene postulates DNA as a repeating

tetranucleotide structure

Rediscovery of Mendels laws byCorrens, von Tschermak, and de Vries

Miescher discovers DNA

Mendel formulates his

fundamental laws of genetics

GENETICS

CYTOLOGY

Morgan and colleagues

develop genetics of Drosophila

Development of

dyes and stains

Virchow: Every cell

comes from a cell

Schleidenand Schwann

formulate cell theory

Brown describes

nuclei

CELL BIOLOGY

Kolliker describes

mitochondria

in muscle cells

Green fluorescent protein used to detect

functional proteins in living cellsDolly the sheep cloned

Mass spectroscopy used to study proteomes

Stereoelectron microscopy used for three-dimensional imagingHuman genome sequenced

Bioinformatics developed to analyze sequence data

Figure 1-1 The Cell Biology Time Line. Although cytology, biochemistry, and genetics began as separate disciplines, they haveincreasingly merged since about the second quarter of the twentieth century.


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prefix cyto- means cell, as does the suffix-cyte.) As wehave already seen, cytology had its origins more than threecenturies ago and depended heavily on the light micro-scope for its initial impetus. The advent of electronmicroscopy and several related optical techniques has led

to considerable additional cytological activity and under-standing.

The second strand represents the contributions ofbiochemistry to our understanding of cellular function.Most of the developments in this field have occurredwithin the last 75 years, though again the roots goback much further. Especially important has been thedevelopment of techniques such as ultracentrifugation,chromatography, and electrophoresis for the separationof cellular components and molecules. The use ofradioactively labeled compounds in the study of enzyme-

catalyzed reactions and metabolic pathways is anothervery significant contribution of biochemistry to ourunderstanding of how cells function. We will encounterthese and other techniques in subsequent chapters as weexplore various aspects of cellular structure and functionand an understanding of relevant techniques becomesnecessary. To locate discussions of specific techniques, seethe Guide to Techniques and Methods inside the frontcover.

The third strand is genetics. Here, the historical con-tinuum stretches back more than 150 years to Gregor

Mendel. Again, however, much of our present under-standing has come within the last 75 years. An especiallyimportant landmark on the genetic strand came with thedemonstration that DNA (deoxyribonucleic acid) is thebearer of genetic information in most life forms, specify-ing the order of subunits, and hence the properties, of theproteins that are responsible for most of the functionaland structural features of cells. Recent accomplishmentson the genetic strand include the sequencing of the entire

genomes (all of the DNA) of humans and other speciesand the cloning(production of genetically identical organ-

isms) of mammals, including sheep, cattle, and cats.To understand present-day cell biology therefore

means to appreciate its diverse roots and the importantcontributions that each of its component strands hasmade to our current understanding of what a cell is andwhat it can do. Each of the three historical strands of cellbiology is discussed briefly here; a fuller appreciation ofeach will come as various aspects of cell structure, func-tion, and genetics are explored in later chapters.

The Cytological Strand Deals with Cellular StructureStrictly speaking, cytology is the study of cells. (Actually,the literal meaning of the Greek word cytos is hollow ves-sel, which fits well with Hookes initial impression ofcells.) Historically, however, cytology has dealt primarilywith cellular structure, mainly through the use of opticaltechniques. Here we describe briefly some of themicroscopy that has been important in cell biology. For amore detailed discussion, see the Appendix.

The Light Microscope. The light microscope was theearliest tool of the cytologists and continues to play animportant role in our elucidation of cellular structure.Lightmicroscopy allowed cytologists to identify membrane-bounded structures such as nuclei, mitochondria, andchloroplasts within a variety of cell types. Such structuresare called organelles (little organs) and are prominentfeatures of most plant and animal (but not bacterial) cells.

Other significant developments include the inventionof the microtome in 1870 and the availability of variousdyes and stains at about the same time. A microtome is aninstrument for slicing thin sections of biological samples,usually after they have been dehydrated and embedded inparaffin or plastic. The technique enables rapid and effi-cient preparation of thin tissue slices of uniform thick-ness. The dyes that came to play so important a role instaining and identifying subcellular structures were devel-oped primarily in the latter half of the nineteenth centuryby German industrial chemists working with coal tarderivatives.

Together with improved optics and more sophisti-cated lenses, these and related developments extendedlight microscopy as far as it could goto the physicallimits of resolution imposed by the wavelengths of visiblelight. As used in microscopy, the limit of resolution refersto how far apart adjacent objects must be in order to bedistinguished as separate entities. For example, to say thatthe limit of resolution of a microscope is 400 nanometers(nm) means that objects need to be at least 400 nm apartto be recognizable as separate entities, whereas a resolu-tion of 200 nm means that objects only 200 nm can bedistinguished from each other. (A nanometer is orone-billionth of a meter; ) The smallerthe limit of resolution, the greater the resolving power ofthe microscope. Expressed in terms of , the wavelength ofthe light used to illuminate the sample, the theoreticallimit of resolution for the light microscope is /2. For visi-ble light in the wavelength range of 400700 nm, the limitof resolution is about 200350 nm. Figure 1-2 illustratesthe useful range of the light microscope and compares itsresolving power with that of the human eye and the elec-tron microscope.

Visualization of Living Cells. The type of microscopydescribed thus far is called brightfield microscopybecausewhite light is passed directly through a specimen that iseither stained or unstained, depending on the structuralfeatures to be examined. A significant limitation of thisapproach is that specimens must be fixed (preserved),

dehydrated, and embedded in paraffin or plastic. Thespecimen is therefore no longer alive, which raises thepossibility that features observed by this method could beartifacts or distortions due to the fixation, dehydration,andembedding processes. To overcome this disadvantage, avariety of special optical techniques have been developedthat make it possible to observe living cells directly. Theseinclude phase-contrast microscopy, differential interferencecontrast microscopy, fluorescence microscopy, confocal

l

l

1 nm=0.001 mm.10:9


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6Chapter 1 A Preview of the Cell

0.1 nm

1 nm

10 nm

100 nm

1 m

10 m

100 m

1 mm

1 cm

0.1 m

1 m

10 m

Human height

Length of some

nerve and

muscle cells

Chicken egg

Frog egg

Eukaryotic cells

Most bacteria

Mitochondrion

Mycoplasma

Viruses

Ribosomes

Proteins

Lipids

Small molecules

Atoms

Nucleus

UNAIDED

EYE

LIGHTMICROSCOPE

ELECTRON

MICROSC

OPE

Figure 1-2 Resolving Power of the Human Eye, the Light Microscope,

and the Electron Microscope. Notice that the vertical axis is on alogarithmic scale to accommodate the range of sizes shown.

microscopy,and digital video microscopy. Table 1-1 depictsthe images seen with each of these techniques andcompares them with the images seen with brightfield

microscopy for both unstained and stained specimens. Eachof these techniques is discussed in the Appendix; here wewill content ourselves with a brief description of each.

Phase-contrast and differential interference contrastmicroscopymake it possible to see living cells clearly (seeTable 1-1). Both of these techniques enhance and amplifyslight changes in the phase of transmitted light as itpasses through a structure that has a different refractiveindex than the surrounding medium. Most modern light

microscopes are equipped for phase-contrast and differ-ential interference contrast in addition to the simpletransmission of light, with conversion from one use to an-other accomplished by interchanging optical components.

Fluorescence microscopyenables researchers to detect

specific proteins or other molecules that are made fluores-cent by coupling them to a fluorescent dye. By the simul-taneous use of two or more such dyes, each coupled to adifferent kind of molecule, the distributions of differentkinds of molecules can be followed in the same cell.

An inherent limitation of fluorescence microscopy isthat the viewer can focus on only a single plane of thespecimen at a given time, yet fluorescent light is emittedthroughout the specimen. As a result, the visible image isblurred by light emitted from regions of the specimenabove and below the focal plane, which historically limited

the technique to flattened cells with minimal depth. Thisproblem is largely overcome by confocal scanning, inwhich a laser beam is used to illuminate a single plane ofthe specimen at a time. This approach gives much betterresolution than traditional fluorescence microscopy whenused with thick specimens such as whole cells. Further-more, the laser beam can be directed to successive focalplanes sequentially, thereby generating a series of imagesthat can be combined to provide a three-dimensional pic-ture of the cell.

Another recent development in light microscopy is

digital video microscopy, which makes use of video cam-eras and computer storage, and allows computerizedimage processing to enhance and analyze images. Attach-ment of a highly light-sensitive video camera to a light mi-croscope makes it possible to observe cells for extendedperiods of time using very low levels of light. This imageintensification is particularly useful for visualizing fluores-cent molecules in living cells with a fluorescence micro-scope.

The Electron Microscope. Despite advances in optical

techniques and contrast enhancement, light microscopy isinevitably subject to the limit of resolution imposed by thewavelength of the light used to view the sample. Even theuse of ultraviolet radiation, with shorter wavelengths,increases the resolution only by a factor of two.

A major breakthrough in resolving power came withthe development of the electron microscope, which wasinvented in Germany in 1932 and came into widespreadbiological use in the early 1950s. In place of visible lightand optical lenses, the electron microscope uses a beam ofelectrons that is deflected and focused by an electromag-

netic field. Because the wavelength of electrons is so muchshorter than that of photons of visible light, the limit ofresolution of the electron microscope is much better thanthat of the light microscope: about 0.10.2 nm for theelectron microscope compared with about 200350 nmfor the light microscope.

However, for biological samples the practical limit ofresolution is usually no better than 2 nm or more, becauseof problems with specimen preparation and contrast.


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Brightfield (unstained specimen):Passes light directly through

specimen; unless cell is naturallypigmented or artificially stained,image has little contrast.

Table 1-1 Different Types of Light Microscopy: A Comparison

Type of Microscopy Light Micrographs of Human Cheek Epithelial Cells Type of Microscopy

Phase contrast: Enhances contrastin unstained cells by amplifying

variations in refractive indexwithin specimen;especially usefulfor examining living,unpigmented cells.

Brightfield (stained specimen):Staining with various dyes enhancescontrast, but most stainingprocedures require that cells be fixed(preserved).

Differential interferencecontrast: Also uses opticalmodifications to exaggeratedifferences in refractive index.

Fluorescence: Shows the locationsof specific molecules in the cell.Fluorescent substances absorbultraviolet radiation and emit visiblelight. The fluorescing molecules mayoccur naturally in the specimen butmore often are made by tagging themolecules of interest withfluorescent dyes or antibodies.

Confocal: Uses lasers and specialoptics to focus illuminating beamon a single plane within thespecimen. Only those regionswithin a narrow depth of focus areimaged. Regions above and belowthe selected plane of view appearblack rather than blurr y.

50 m

Source: From Campbell and Reece, Biology6th

edition (San Francisco: Benjamin Cummings,2002), p. 110.

Nevertheless, the electron microscope has about 100 timesmore resolving power than the light microscope (seeFigure 1-2). As a result, the useful magnification is alsogreater: up to 100,000-fold for the electron microscope,compared with about 1000- to 1500-fold for the light mi-croscope.

Electron microscopes are of two basic designs: the

transmission electron microscope (TEM) and thescanning electron microscope (SEM). Both are describedin detail in the Appendix. Transmission and scanningelectron microscopes are similar in that each employs abeam of electrons, but they use quite different mecha-nisms to form the image. As the name implies, a TEMforms an image from electrons that are transmittedthrough the specimen. An SEM, on the other hand, scansthe surface of the specimen and forms an image by detect-ing electrons that are deflected from the outer surface ofthe specimen. Scanning electron microscopy is an espe-

cially spectacular technique because of the sense of depthit gives to biological structures (Figure 1-3). Most of theelectron micrographs in this book were obtained by theuse of either a TEM or an SEM and are identified as suchby the appropriate three-letter abbreviation at the end ofthe figure legend.

Because of the low penetration power of electrons,samples prepared for electron microscopy must be ex-ceedingly thin. The instrument used for this purpose is

called an ultramicrotome . It is equipped with a diamondknife and can cut sections as thin as 20 nm. Substantiallythicker samples can also be examined by electron micros-copy, but a much higher accelerating voltage is then re-quired to increase the penetration power of the electronsadequately. Such a high-voltage electron microscope usesaccelerating voltages up to several thousand kilovolts

(kV), compared with the range of 50100 kV common tomost conventional instruments. Sections up to thickcan be studied with such a high-voltage instrument. Thisthickness allows organelles and other cellular structures tobe examined in more depth.

Several specialized techniques of electron microscopyare in use; each is just an alternative way of preparingsamples for transmission electron microscopy. These in-clude negative staining, shadowing, freeze fracturing, and

freeze etching, each of which is a useful means of visualiz-ing specimens in three dimensions. Also valuable for this

purpose is a technique called stereo-electron microscopy, inwhich the same sample is photographed at two slightlydifferent angles using a special specimen stage that can betilted relative to the electron beam. These techniques aredescribed in detail in the Appendix.

Electron microscopy has revolutionized our under-standing of cellular architecture by making detailed ultra-structural investigations possible. Some organelles (suchas nuclei or mitochondria) are large enough to be seen

1 mm


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8Chapter 1 A Preview of the Cell

(a) Human neuroblastoma cells (b) Pollen grain50 m 10 m

Figure 1-3 Scanning Electron Microscopy. A scanning electron microscope was used to visualize (a) cultured humanneuroblastoma cells and (b) a pollen grain.

with a light microscope but can be studied in much

greater detail with an electron microscope. In addition,electron microscopy has revealed cellular structures thatare too small to be seen with a light microscope. These in-clude ribosomes, membranes, microtubules, and microfil-aments (see Figure 1A-2 on p. 3).

The Biochemical Strand Covers the Chemistryof Biological Structure and Function

At about the time when cytologists were starting to ex-plore cellular structure with their microscopes, other

scientists were making observations that began to ex-plain and clarify cellular function. Much of what is nowcalled biochemistry dates from a discovery reported bythe German chemist Friedrich Whler in 1828. Whlerwas a contemporary (as well as fellow countryman) ofSchleiden and Schwann. He revolutionized our thinkingabout biology and chemistry by demonstrating thaturea, an organic compound of biological origin, couldbe synthesized in the laboratory from an inorganicstarting material, ammonium cyanate. Until then, it hadbeen widely held that living organisms were a world

unto themselves, not governed by the laws of chemistryand physics that apply to the nonliving world. By show-ing that a compound made by living organismsa bio-chemicalcould be synthesized in a laboratory justlike any other chemical, Whler helped to break downthe conceptual distinction between the living and non-living worlds and to dispel the notion that biochemicalprocesses were somehow exempt from the laws of chem-istry and physics.

Another major advance came about 40 years later,

when Louis Pasteur linked the activity of living organismsto specific processes by showing that living yeast cells wereneeded to carry out the fermentation of sugar into alco-hol. This observation was followed in 1897 by the findingof Eduard and Hans Buchner that fermentation could alsotake place with extracts from yeast cellsthat is, the intactcells themselves were not required. Initially, such extractswere called ferments, but gradually it became clear thatthe active agents in the extracts were specific biologicalcatalysts that have since come to be called enzymes.

Significant progress in our understanding of cellular

function came in the 1920s and 1930s as the biochemicalpathways for fermentation and related cellular processeswere elucidated. This was a period dominated by Germanbiochemists such as Gustav Embden, Otto Meyerhof, OttoWarburg, and Hans Krebs. Several of these men have longsince been immortalized by the pathways that bear theirnames. For example, the Embden-Meyerhof pathway forglycolysis was a major research triumph of the early 1930s.It was followed shortly by the Krebs cycle (also known asthe TCA cycle). Both of these pathways are importantbecause of their role in the process by which cells extract

energy from foodstuffs. At about the same time, FritzLipmann, an American biochemist, showed that the high-energy compound adenosine triphosphate (ATP) is theprincipal energy storage compound in most cells.

An important advance in the study of biochemicalreactions and pathways came as radioactive isotopes suchas and began to be used to trace the metabolicfate of specific atoms and molecules. (As you may recallfrom chemistry, different atoms of a given chemical

32P3H, 14C,


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The Emergence of Modern Cell Biology9

element may have the same atomic number and nearlyidentical properties but differ in the number of neutronsand hence in atomic weight; an isotope refers to the atomswith a specific number of neutrons and thus a particularatomic weight. A radioactive isotope, or radioisotope, is an

isotope that is unstable, emitting subatomic particles[either alpha or beta particles] and, in some cases, gammarays as it undergoes spontaneous conversion to a stableform.) Melvin Calvin and his colleagues at the Universityof California, Berkeley, were pioneers in this field as theytraced the fate of carbon dioxide, inilluminated algal cells that were actively photosynthesiz-ing. Their work, carried out in the late 1940s and early1950s, led to the elucidation of the Calvin cycle, as themost common pathway for photosynthetic carbon metab-olism is called. The Calvin cycle was the first metabolic

pathway to be elucidated using a radioisotope.Biochemistry took another major step forward with

the development of centrifugation as a means of separat-ing and isolating subcellular structures and macromole-cules on the basis of size, shape, and/or density, a processcalled subcellular fractionation. Centrifugation tech-niques used for this purpose include differential centrifu-

gation and density gradient centrifugation, which separateorganelles and other subcellular structures on the basis ofsize and/or density differences, and equilibrium densitycentrifugation, a powerful technique for resolving or-

ganelles and macromolecules based on density differ-ences. Each of these techniques is described in detail onBox 12A on pp. 322326. Especially useful for the resolu-tion of small organelles and macromolecules is theultracentrifuge, which was developed in Sweden byTheodor Svedberg in the late 1920s. An ultracentrifuge iscapable of very high speedsover 100,000 rpmand canthereby subject samples to forces exceeding 500,000 timesthe force of gravity (g). In many ways, the ultracentrifugeis as significant to biochemistry as the electron micro-scope is to cytology. In fact, both instruments were devel-

oped at about the same time, so the ability to seeorganelles and other subcellular structures came almostsimultaneously with the capability to isolate and purifythem.

Other biochemical techniques that have proven veryuseful for the isolation and purification of subcellularcomponents include chromatography and electrophore-sis. Chromatography is a general term that includes a va-riety of techniques in which a mixture of molecules insolution is progressively fractionated as the solution flowsover a nonmobile absorbing phase, usually contained in a

column. Chromatographic techniques separate moleculeson the basis of size, charge, or affinity for specific mole-cules or functional groups. An example of a chromato-graphic technique is shown in Figure 7-9 on p. 165.

Electrophoresis refers to several related techniquesthat utilize an electrical field to separate molecules basedon their mobility. The rate at which any given moleculemoves during electrophoresis depends upon its charge andits size. The most common medium for electrophoretic

14CO2,14C-labeled

separation of proteins and nucleic acids is a gel of eitherpolyacrylamide or agarose. The use of polyacrylamide gelelectrophoresis for the resolution of proteins is illustratedin Figure 7-22 on p. 177.

With an enhanced ability to see subcellular structures,

to fractionate, and to isolate them, cytologists and bio-chemists began to realize the extent to which their respec-tive observations on cellular structure and function couldcomplement each other, thereby laying the foundationsfor modern cell biology.

The Genetic Strand Focuses on Information Flow

The third strand in the cord of cell biology is genetics. Likethe other two, this strand has important roots in the nine-teenth century. In this case, the strand begins with Gregor

Mendel, whose studies with the pea plants he grew in amonastery garden must surely rank among the most fa-mous experiments in all of biology. His findings werepublished in 1866, laying out the principles of segregationand independent assortment of the hereditary factorsthat we know today as genes. These were singularly im-portant principles, destined to provide the foundation forwhat would eventually be known as Mendelian genetics.But Mendel was clearly a man ahead of his time. His workwent almost unnoticed when it was first published andwas not fully appreciated until its rediscovery nearly

35 years later.As a prelude to that rediscovery, the role of the nu-

cleus in the genetic continuity of cells came to be appreci-ated in the decade following Mendels work. In 1880,Walther Flemming identified chromosomes, threadlikebodies seen in dividing cells. Flemming called the divisionprocess mitosis, from the Greek word for thread. Chromo-some number soon came to be recognized as a distinctivecharacteristic of a species and was shown to remain con-stant from generation to generation. That the chromo-somes themselves might be the actual bearers of genetic

information was suggested by Wilhelm Roux as early as1883 and was expressed more formally by August Weiss-man shortly thereafter.

With the roles of the nucleus and chromosomes es-tablished and appreciated, the stage was set for the redis-covery of Mendels initial observations. This came in 1900,when his studies were cited almost simultaneously bythree plant geneticists working independently: CarlCorrens in Germany, Ernst von Tschermak in Austria, andHugo de Vries in Holland. Within three years, thechromosome theory of hereditywas formulated by Wal-

ter Sutton, who was the first to link the chromosomalthreads of Flemming with the hereditary factors ofMendel.

Suttons theory proposed that the hereditary factorsresponsible for Mendelian inheritance are located on thechromosomes within the nucleus. This hypothesis re-ceived its strongest confirmation from the work ofThomas Hunt Morgan and his students at ColumbiaUniversity during the first two decades of the twentieth


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century. They chose Drosophila melanogaster, the com-mon fruit fly, as their experimental species. By identifyinga variety of morphological mutants ofDrosophila, Mor-gan and his co-workers were able to link specific traits tospecific chromosomes.

Meanwhile, the foundation for our understanding ofthe chemical basis of inheritance was also slowly beinglaid. An important milestone was the discovery ofDNA byJohann Friedrich Miescher in 1869. Using such unlikelysources as salmon sperm and human pus from surgical

bandages, Miescher isolated and described what he callednuclein. But, like Mendel, Miescher was ahead of histime. It was about 75 years before the role of his nuclein asthe genetic information of the cell came to be fully appre-ciated.

As early as 1914, DNA was implicated as an importantcomponent of chromosomes by Robert Feulgens stainingtechnique, a method that is still in use today. But littleconsideration was given to the possibility that DNA could

be the bearer of genetic information. In fact, that was con-sidered quite unlikely in light of the apparently uninter-esting structure of the monomer constituents of DNA(called nucleotides) that were known by 1930. Until themiddle of the twentieth century, it was widely held thatgenes were made up of proteins, since these were the onlynuclear components that seemed to account for the obvi-ous diversity of genes.

A landmark experiment that clearly pointed to DNAas the genetic material was reported in 1944 by Oswald

Avery, Colin MacLeod, and Maclyn McCarty. Their workfocused on the phenomenon of genetic transformation inbacteria, to be discussed in Chapter 18. Their evidence wascompelling, but the scientific community remainedlargely unconvinced of the conclusion. Just eight yearslater, however, a considerably more favorable receptionwas accorded the report of Alfred Hershey and MarthaChase that DNA, and not protein, enters a bacterial cellwhen it is infected by a bacterial virus.

If asked what they expect to get out of a science textbook, mostreaders would probably reply that they intend to learn the facts

relevant to the particular scientific area the book is aboutcellbiology, in the case of the text you are reading right now. Ifpressed to explain what a fact is, most people would probablyreply that a fact is something that we know to be true. Thatsense of the word agrees with the dictionary, since one of thedefinitions offactis a piece of information presented as havingobjective reality.

To a scientist, however, a fact is a much more tenuous pieceof information than such a definition might imply. The factsof science are really just attempts to state our current under-standing of the natural world around us, based on observationsthat we make and experiments that we do. As such, a given

fact is only as sound as the observations or experiments onwhich it is based and can be modified or superseded at any timeby a better understanding based on more careful observationsor more discriminating experiments. As one scientist so aptlyput it, truth to a researcher is not a citadel of certainty to bedefended against error; it is a shady spot where one eats lunchbefore tramping on(White, 1968, p. 3).

Cell biology is rich with examples of facts that were oncewidely held but have since been superseded as cell biologistshave tramped onto a better understanding of the phenomenathose facts attempted to explain. As recently as the early nine-teenth century, for example, it was widely held (i.e., regarded as

fact) that living matter consisted of substances quite differentfrom those in nonliving matter. According to this view, calledvitalism, the chemical reactions that occurred within living mat-ter did not follow the known laws of chemistry and physics butwere instead directed by a vital force. Then came FriedrichWhlers demonstration (in 1828) that the biological com-pound urea could be synthesized in the laboratory from aninorganic compound, thereby undermining one of the facts ofvitalism. The other fact was refuted by the work of Eduard

and Hans Buchner, who showed (in 1897) that nonlivingextracts from yeast cells could ferment sugar into ethanol. Thus,

a view held as fact by generations of scientists was eventuallydiscredited and replaced by the new fact that the componentsand reactions of living matter are not a world unto themselves,but follow all the laws of chemistry and physics.

For a more contemporary example, consider what we knowabout the energy needed to support life. Until recently, it wasregarded as a fact that the sun is the ultimate source of allenergy in the biosphere, such that every organism either usessolar energy directly (i.e., green plants, algae, and certain bacte-ria) or is a part of a food chain that is sustained by such photo-synthetic organisms. Then came the discovery ofdeep-seathermal vents and the thriving communities of organisms that

live around them, none of which depends on solar energy.Instead, these organisms depend on the bond energy of hydro-gen sulfide which is extracted by bacteria that livearound the thermal vents and is used to synthesize organiccompounds from carbon dioxide. These bacteria form the basisof food chains that include zooplankton (microscopic animals),worms, and other residents of the thermal vent environment.

Thus, the facts presented in biology textbooks such as thisone are nothing more than our best current attempts todescribe and explain the workings of the biological worldaround us. They are subject to change whenever we becomeaware of new or better information.

How does new and better information become available?Scientists usually assess new information with a systematicapproach called the scientific method. As Figure 1B-1 indicates,the scientific method begins as a researcher makes observations,either in the field or in a research laboratory. Based on theseobservations and on knowledge gained in prior studies, the sci-entist formulates a testable hypothesis, a tentative explanation ormodel consistent with the observations and with prior knowl-edge that can be tested experimentally. Next, the investigator

(H2S),

Biology,Facts, and the Scientific Method

Further InsightsB o x 1 B


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The Emergence of Modern Cell Biology11

Meanwhile, George Beadle and Edward Tatum, work-ing in the 1940s with the bread mold Neurospora crassa,formulated the one geneone enzyme concept, assert-ing that the function of a gene is to control the productionof a single, specific protein. Shortly thereafter, in 1953,James Watson and Francis Crick proposed their now-famous double helix model for DNA structure, with fea-tures that immediately suggested how replication andgenetic mutations could occur. Thereafter, the features ofDNA function fell rapidly into place, establishing thatDNA

specifies the order of monomers (amino acids) and hencethe properties of proteins and that several different kindsof RNA (ribonucleic acid) molecules serve as intermedi-ates in protein synthesis.

The 1960s brought especially significant develop-ments, including the discovery of the enzymes thatsynthesize DNA and RNA (DNA polymerases and RNApolymerases, respectively) and the crackingof the geneticcode, which specifies the relationship between the order of

nucleotides in a DNA (or RNA) molecule and the order ofamino acids in a protein. At about the same time, JacquesMonod and Franois Jacob deduced the mechanism re-sponsible for the regulation of bacterial gene expression.

Important techniques along the genetic strand ofFigure 1-1 include the separation ofDNA molecules andfragments by ultracentrifugation and gel electrophoresis.Of equal, if not greater, importance is nucleic acidhybridization, which includes a variety of related tech-niques that depend on the ability of two single-stranded

nucleic acid molecules with complementary base se-quences to bind, or hybridize, to each other, thereby form-ing a double-stranded hybrid. These techniques can beapplied to DNA-DNA, DNA-RNA, and even RNA-RNA in-teractions, and they are very useful for the isolation ofspecific DNA or RNA molecules or fragments thereof.

The technological advance that has unquestionablycontributed the most to our understanding of geneexpression is the development of recombinant DNA

designs a controlled experimentto test the hypothesis by varying

specific conditions while holding everything else as constant aspossible. The scientist then collects the data, interprets the results,and draws reasonable conclusions, which obviously must be con-sistent not only with the results of this particular experimentbut with prior knowledge as well.

To a practicing scientist, the scientific method is more a wayof thinking than a set of procedures to be followed. Most likely,this is the way our ancestors explained and interpreted naturalphenomena long before scientists were trained at universities

and long before students read essays about the scientificmethod!

When illustrated by a diagram such as Figure 1B-1, the sci-entific method looks very neat and orderly. Not all scientific dis-coveries are made in this way, however. Many importantadvances in biology have come about more by accident than byplan. Alexander Flemings discovery of penicillin in 1928 is aclassic example. Fleming, a Scottish physician andbacteriologist, accidentally left a culture dish ofStaphylococcusbacteria uncovered, such that it was inadvertently exposed tocontamination by other microorganisms. Fleming was about todiscard the contaminated culture when he happened to noticesome clear patches where the bacteria were not growing. Rea-soning that the bacterial growth may have been inhibited by

some contaminant in the air and recognizing how important aninhibitor of bacterial growth might be, Fleming kept the culturedish and began attempts to isolate and characterize the sub-stance. The actual identification of penicillin and the demon-stration that it was the product of a mold was left to others, butFleming is credited with the initial discovery.

Boxes in subsequent chapters will acquaint you with furtherexamples of apparently accidental discoveries. Regardless ofhow accidental such discoveries may appear, however, it isalmost always true that chance favors the prepared mind.Behind the apparent chanceof each such discovery is theprepared mind that has been trained to observe carefully and

to think astutely.As you proceed through this text, be on the outlook forapplications of the scientific method. You will find that regard-less of the approach, the conclusions from each experiment addto our knowledge of how biological systems work and usuallylead to more questions as well, continuing the cycle of scientificinquiry.And thats good news if you aspire to a career inresearch, because its your best insurance that there will still bequestions to answer when you are ready to begin.

Figure 1B-1 The Scientific Method.

Make initial observations1

Formulate a testable hypothesis2

Design a controlled experiment3

Collect data4

Interpret results5

Draw reasonable conclusions6

Consult prior

knowledge


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technologyin the 1970s. This technology was made possi-ble by the discovery ofrestriction enzymes. These enzymeshave the ability to cleave DNA molecules at specific se-quences called restriction sites, which makes them power-ful tools for cutting large DNA molecules into smaller

restriction fragments that can be recombined in variousways. Using these enzymes, scientists can createrecombinant DNA molecules containing DNA sequencesfrom two different sources. This capability led quickly tothe development ofgene cloning, a process for generatingmany copies of specific DNA sequences. These techniquesare explained and explored in detail in Chapters 18 and 20.

At about the same time, methods were devised forrapidly determining the base sequences of DNA fragments.The importance ofDNA sequencingtechnology can hardlybe overestimated. In fact, the technology is now so com-

monplace and automated that it is routinely applied notjust to individual genes but to entiregenomes (that is thetotal DNA content of a cell). Initially, genome sequencingwas applied mainly to bacterial genomes because they arerelatively smalla few million bases,typically. But DNA se-quencing has long since been successfully applied to muchlarger genomes, including those from species of yeast,roundworm, plants, and animals that are of special interestto researchers. The ultimate triumph was the sequencing ofthe entire human genome, which contains about 3.2 billionbases. This feat was accomplished by the Human Genome

Project, a cooperative international effort that began in1990, involved hundreds of scientists, and established thecomplete sequence of the human genome by 2003.

The challenge of analyzing the vast amount of datagenerated byDNA sequencing has led to a new discipline,called bioinformatics, which merges computer scienceand biology as a means of making sense of sequence data.In the case of the human genome, this approach has led tothe recognition that there are at least 35,000 protein-coding genes in the human genome, about half of whichwere not known to exist prior to genome sequencing.

With the DNA sequences for these genes now known,scientists are beginning to look beyond the genome tostudy theproteome, which encompasses the structure andproperties of every protein produced by a genome.

These and other techniques helped to launch an era ofmolecular genetics that continues to revolutionize biol-ogy. In the process, the historical strand of genetics thatdates back to Mendel became intimately entwined withthose of cytology and biochemistry, and the discipline ofcell biology as we know it today came into being.

Facts and the Scientific Method

To become familiar with an area of science such as cellbiology means, at least in part, to learn thefacts about thatsubject. Even in this short introductory chapter, we havealready encountered a number of facts about cell biology.When we say, for example, that all organisms consist ofone or more cellsor that DNA is the bearer of genetic in-formation, we recognize these statements as facts of cell

biology. But we also recognize that the first of these state-ments was initially regarded as part of a theory and thesecond statement actually replaced an earlier misconcep-tion that genes were made of proteins.

Clearly, then, a scientific fact is a much more tenu-

ous piece of information than our everyday sense of theword might imply. To a scientist, a fact is simply an at-tempt to state our best current understanding of a specificphenomenon and is only valid until it is revised or re-placed by a better understanding. Box 1B explores themeaning of facts in biology and the scientific methodby which new and better information becomes available.

As we consider the scientific method, we need to rec-ognize several important terms that scientists use to indi-cate the degree of certainty with which a specificexplanation or concept is regarded as true. Three terms

are especially significant: hypothesis, theory, and law.Of the three, hypothesis is the most tentative. A

hypothesis is simply a statement or explanation that isconsistent with most of the observational and experimen-tal evidence to date. Suppose, for example, that you haveexperienced heartburn three times in the last month andthat you had in each case eaten a pepperoni pizza shortlybefore experiencing the heartburn. A reasonable hypothe-sis might be that the heartburn is somehow linked to theconsumption of pepperoni pizza. Often, a hypothesistakes the form of a modelthat appears to provide a reason-

able explanation of the phenomenon in question.To be useful to scientists, a hypothesis must be

testable that is, it must be possible to design a controlledexperiment that will either confirm or discredit thehypothesis. Based on initial observations and priorknowledge (from the work of other investigators, mostlikely), the scientist formulates a testable hypothesis andthen designs a controlled experiment to determinewhether or not the hypothesis will be supported by dataor observations (see Figure 1B-1).

When a hypothesis or model has been tested critically

under many different conditions usually by many differ-ent investigators using a variety of approaches and isconsistently supported by the evidence, it gradually ac-quires the status of a theory. By the time an explanation ormodel comes to be regarded as a theory, it is generally andwidely accepted by most scientists in the field. The celltheorydescribed earlier in this chapter is an excellent ex-ample. There is little or no dissent or disagreement amongbiologists concerning its three tenets. Two more recent ex-planations that have acquired the status of theory are thechemiosmotic modelthat explains how mitochondrial ATP

production is driven by an electrochemical proton gradi-ent across the inner mitochondrial membrane (discussedin Chapter 10), and the fluid mosaic modelof membranestructure that we will encounter in Chapter 7.

When a theory has been so thoroughly tested andconfirmed over a long period of time by a large number ofinvestigators that virtually no doubt remains whatever, itmay eventually come to be regarded as a law. The law of

gravity comes readily to mind, as do the several laws of


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Problem Set 13

Problem Set

More challenging problems are marked with a .

1-1 The Historical Strands of Cell Biology. For each of thefollowing events, indicate whether it belongs mainly to the cyto-logical (C), biochemical (B),or genetic (G) strand in the histor-

ical development of cell biology.(a) Kllicker describes sarcosomes(now called mitochon-

dria) in muscle cells (1857).

(b) HoppeSeyler isolates the protein hemoglobin in crys-talline form (1864).

(c) Haeckel postulates that the nucleus is responsible forheredity (1868).

(d) Ostwald proves that enzymes are catalysts (1893).

(e) Muller discovers that X-rays induce mutations (1927).

(f) Davson and Danielli postulate a model for the structure ofcell membranes (1935).

(g) Beadle and Tatum formulate the one geneone enzymehypothesis (1940).

(h) Claude isolates the first mitochondrial fractions from ratliver (1940).

(i) Lipmann postulates the central importance of ATP in cel-lular energy transactions (1940).

(j) Avery, MacLeod,and McCarty demonstrate that bacterialtransformation is attributable to DNA, not protein (1944).

(k) Palade, Porter, and Sjstrand each develop techniques forfixing and sectioning biological tissue for electron

microscopy (19521953).(l) Lehninger demonstrates that oxidative phosphorylation

depends for its immediate energy source on the transportof electrons in the mitochondrion (1957).

1-2 Cell Sizes. To appreciate the differences in cell size illus-trated in Figure 1A-1 on p. 2, consider these specific examples.Escherichia coli, a typical bacterial cell, is cylindrical in shape,with a diameter of about and a length of about As atypical animal cell, consider a human liver cell, which is roughly

2 mm.1 mm

spherical in shape and has a diameter of about And for atypical plant cell, consider the columnarpalisade cells locatedjust beneath the upper surface of many plant leaves. These cellsare cylindrical in shape, with a diameter of about and alength of about

(a) Calculate the approximate volume of each of these threecell types in cubic micrometers. (Recall that for acylinder and that for a sphere.)

(b) Approximately how many bacterial cells would fit in theinternal volume of a human liver cell?

(c) Approximately how many liver cells would fit inside a pal-isade cell?

1-3 Sizing Things Up. To appreciate the sizes of the subcellu-lar structures shown in Figure 1A-2 on p. 3, consider the

following calculations:(a) All cells and many subcellular structures are surrounded by

a membrane. Assuming a typical membrane to be about8 nm wide, how many such membranes would have to bealigned side by side before the structure could be seen withthe light microscope? How many with the electron micro-scope?

(b) Ribosomes are the structures in cells on which the processof protein synthesis takes place. A human ribosome is aroughly spherical structure with a diameter of about30 nm. How many ribosomes would fit in the internalvolume of the human liver cell described in Problem 1-2 ifthe entire volume of the cell were filled with ribosomes?

(c) The genetic material of the Escherichia coli cell described inProblem 1-2 consists of a DNA molecule with a diameterof 2 nm and a total length of 1.36 mm. (The molecule isactually circular, with 1.36 mm as its circumference.) To beaccommodated in a cell that is only a few micrometerslong, this large DNA molecule is tightly coiled and foldedinto a nucleoidthat occupies a small proportion of theinternal volume of the cell. Calculate the smallest possible

V=4pr3/3V=pr2h

35 mm.20 mm

20 mm.

thermodynamics that we will encounter in Chapter 5. Youmay also be familiar with Ficks law of diffusion, the ideal

gas laws, and other concepts from physics and chemistrythat are generally regarded as laws. Some of the most no-table biological examples are from geneticsMendels

laws of heredityand the Hardy Weinberg law, for instance.In general, however, biologists are quite conservative with

the term. Even after more than 150 years, the cell theory isstill regarded as just that a theory. Perhaps our reluc-tance to label explanations of biological phenomena aslaws is a reflection of the great diversity of life forms andthe consequent difficulty we have convincing ourselves

that we will never find organisms or cells that are excep-tions to even our most well-documented theories.

The biological world is a world of cells. Allliving organisms are made up of one ormore cells, each of which came from a pre-existing cell. Although the importance ofcells in biological organization has beenappreciated for about 150 years, the

discipline of cell biology as we know ittoday is of much more recent origin.Modern cell biology has come about bythe interweaving of three historically

distinct strandscytology, biochemistry,and geneticswhich in their early devel-

opment probably did not seem at allrelated. But the contemporary cell biolo-gist must understand all three strands,because they complement one another in

the quest to learn what cells are and howthey function.

Perspective

A Preview of the Cell

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