Classic Experiments From Lodish

3.1

Bringing an Enzyme Back to Life

By the 1950s, scientists realized that DNA held the code that allowed

proteins to be synthesized. Nevertheless, how a chain of amino

acids folds into a fully functional protein, with the proper three-

dimensional structure, remained a mystery. A mechanism must exist

to assure the proper folding of the protein. But where did that

information come from? In 1957, Christian Anfinsen published the first

evidence that the information for proper folding was held within the

protein itself.

BackgroundProteins are made from combinations of 20 amino acids that

then fold into complex structures. The unfolded amino acid

chain is called the primary structure. To have biological ac-

tivity, the protein must fold into proper secondary and ter-

tiary structures. These structures are held together by chem-

ical interactions between the side chains of the amino acids,

including hydrogen bonds, hydrophobic interactions, and,

at times, covalent bonds. How these higher structures form

has long been a mystery. Does the protein fold correctly as

it is synthesized or does it require the action of other pro-

teins to correctly fold it? Can it correctly fold on its own

spontaneously?

In the 1950s, Anfinsen was a biochemist interested in

the proper folding of proteins. Specifically, he was investi-

gating the formation of disulfide bridges, which are cova-

lent bonds between cysteine side chains that serve as one of

the major anchors holding together the structure of secreted

proteins. He believed that the protein itself contained all the

information necessary for proper protein folding. He pro-

posed the “thermodynamic hypothesis,” which stated that

the biologically active structure of a protein was also the

most thermodynamically stable under in vivo conditions. In

other words, if the intracellular conditions could be mimic-

ked in a test tube, then a protein would naturally fold into

its active conformation. He began his work on a secreted

enzyme, bovine pancreatic ribonuclease, and studied its abil-

ity to properly fold outside of the cell.

The ExperimentProteins perform a wide variety of functions in the cell. Re-

gardless of its function, a protein must be properly folded

to carry out its biological role. For protein folding studies

it is best to study an enzyme whose biological activity can

be easily monitored by performing in vitro. Anfinsen chose

a small, secreted protein, the enzyme ribonuclease, in which

he could monitor proper folding by assaying its ability to

catalyze the cleavage of RNA.

Ribonuclease, a secreted protein, is active under oxidiz-

ing conditions in vitro. The tertiary structure of active ri-

bonuclease is held together by four disulfide bridges. Adding

a reducing agent, which reduces the disulfide bond between

two cysteine side chains to two free sulfhydryl groups, can

disrupt this covalent interaction. Complete denaturation of

ribonuclease requires treatment with a reducing agent.

Anfinsen monitored the reduction of ribonuclease by mea-

suring the number of free sulfhydryl groups present in the

Classic Experiment

TABLE 3-1 Cell-free Refolding of Ribonuclease

Activity as a Percent of

Concentration of Protein Equivalent Concentration

(mg/ml) of Native Ribonuclease

7.0 31%

4.8 70%

2.3 75%

0.9 77%

0.35 94%

[Data adapted from C. B. Anfinsen and E. Haber, 1961, Journal of Biological Chemistry 236:1362.]

protein. In the oxidized state, there are no free sulfhydryl

groups in ribonuclease because each cysteine residue is in-

volved in a disulfide bond. In the completely reduced state,

on the other hand, ribonuclease contains eight free

sulfhydryl groups. Anfinsen exploited this difference to as-

sess the extent of reduction by using spectrophotometric as-

say to titrate the number of sulfhydryl groups.

To study protein folding outside the cell, one must first

denature the protein. Proteins are easily denatured by heat,

mechanical disruption such as shaking, and chemical treat-

ment. Proteins with disulfide bridges require an additional

measure of treatment with a reducing agent to break apart

these covalent bonds. To denature ribonuclease, Anfinsen

first reduced the disulfide bridges with thioglycolic acid. He

then denatured the protein by using a high concentration of

urea and incubating the solution at room temperature. He

demonstrated that this treatment rendered the enzyme in-

active by showing that ribonuclease was now unable to cat-

alyze the cleavage of RNA. Using the spectrophotometric

assay, he went on to show that the inactive ribonuclease con-

tained eight sulfhydryl groups, which corresponded to the

four broken disulfide bridges. With a completely reduced,

denatured protein in hand, Anfinsen then could ask: Can a

denatured enzyme correctly fold in vitro and become active

again?

To find the answer, Anfinsen allowed a solution of re-

duced, denatured ribonuclease to oxidize. He removed the

urea from the denatured enzyme by precipitation. Next, he

resuspended the urea-free denatured ribonuclease in a

buffered solution and incubated it for two to three days. Ex-

posure to molecular oxygen in the atmosphere oxidized the

cysteine residues. He then compared the activity of this re-

natured ribonuclease to that of the native enzyme. In initial

experiments, 12–19 percent of the previously inactive pro-

tein were able to catalyze the cleavage of RNA once again.

Proteins aggregate at high concentrations, which makes it

difficult for them to fold properly. By decreasing the over-

all concentration of ribonuclease in solution, Anfinsen

showed that up to 94 percent of the protein could be re-

folded (see Table 3.1). The enzyme had folded back to its

active conformation outside of the cell, demonstrating that

the information for the protein folding is contained in the

protein itself.

DiscussionThrough careful experiments, Anfinsen demonstrated that

the information required to properly fold a protein is con-

tained in its primary sequence. His careful analysis of the

chemistry of this process answered a fundamental question

in biology. He went on to demonstrate the cell-free refold-

ing of other enzymes, including proteins lacking disulfide

bridges. While it is possible to properly fold a number of

proteins outside of the normal protein-processing machin-

ery in the cell, this process is greatly accelerated in vivo by

a number of enzymes. Anfinsen continued to study the

protein-folding problem. Although the “thermodynamic

hypothesis” does not hold true for all proteins, Anfinsen’s

demonstration of the cell-free refolding of ribonuclease made

a mark on the field of biochemistry. In 1972, he received

the Nobel Prize for Chemistry for his work.

BackgroundProteins are made from combinations of 20 differentamino acids. The genes that encode proteins—that is, spec-ify the type and linear order of their component aminoacids—are located in DNA, a polymer made up of onlyfour different nucleotides. The DNA code is transcribedinto RNA, which is also composed of four nucleotides.Nirenberg’s studies were premised on the hypothesis thatthe nucleotides in RNA form “codewords,” each of whichcorresponds to one of the amino acids found in protein.During protein synthesis, these codewords are translatedinto a functional protein. Thus, to understand how DNAdirects protein synthesis, Nirenberg set out to understandthe relationship between RNA codewords and protein synthesis.

At the outset of his studies, much was already knownabout the process of protein synthesis, which occurs onribosomes. These large ribonuleoprotein complexes canbind two different types of RNA: messenger RNA(mRNA), which carries the exact protein-specifying codefrom DNA to ribosomes, and smaller RNA molecules nowknown as transfer RNA (tRNA), which deliver aminoacids to ribosomes. tRNAs exist in two forms: those thatare covalently attached to a single amino acid, known asamino-acylated or “charged” tRNAs, and those that haveno amino acid attached called “uncharged” tRNAs. Afterbinding of the mRNA and the amino-acylated tRNA to

CRACKING THE GENETIC CODE

By the early 1960s molecular biologists had adopted the so-called “central

dogma,” which states that DNA directs synthesis of RNA (transcription),

which then directs assembly of proteins (translation). However, researchers still

did not completely understand how the “code” embodied in DNA and subse-

quently in RNA directs protein synthesis. To elucidate this process, Marshall

Nirenberg embarked upon a series of studies that would lead to solution of the

genetic code.

4.1Classic Experiment

the ribosome, a peptide bond forms between the aminoacids, beginning protein synthesis. The nascent proteinchain is elongated by the subsequent binding of additionaltRNAs and formation of a peptide bond between theincoming amino acid and the end of the growing chain.Although this general process was understood, the ques-tion remained: How does the mRNA direct protein synthesis?

When attempting to address complex processes such asprotein synthesis, scientists divide large questions into aseries of smaller, more easily addressed questions. Prior toNirenberg’s study, it had been shown that when phenyl-alanie charged tRNA was incubated with ribosomes andpolyuridylic acid (polyU), peptides consisting of onlyphenylalanine were produced. This finding suggested thatthe mRNA codeword, or codon, for phenylalanine ismade up of the nucleosides containing the base uracil.Similar studies with polycytadylic acid (polyrC) andpolyadenylic acid (polyrA) showed these nucleosides con-taining the bases cytadine and adenine made up thecodons for proline and lysine, respectively. With thisknowledge in hand, Nirenberg asked the question: Whatis the minimum chain length required for tRNA binding toribosomes? The system he developed to answer this ques-tion would give him the means to determine which amino-acylated tRNA would bind which m-RNA codon, effec-tively cracking the genetic code.

8945d_003-012 6/18/03 11:04 AM Page 3 mac85 Mac 85:1st shift: 1268_tm:8945d:

The ExperimentThe first step in determining the minimum length ofmRNA required for tRNA recognition was to develop anassay that would detect this interaction. Since previousstudies had shown that ribosomes bind mRNA and tRNAsimultaneously, Nirenberg reasoned that ribosomes couldbe used as a bridge between a known mRNA codon and aknown tRNA. When the three components of protein syn-thesis are incubated together in vitro, they should form acomplex. After devising a method to detect this complex,Nirenberg could then alter the size of the mRNA to deter-mine the minimum chain length required for tRNA recognition.

Before he could begin his experiment, Nirenberg neededboth a means to separate the complex from unbound components and a method to detect tRNA binding to theribosome. To isolate the complex he exploited the abilityof nylon filters to bind large RNA molecules, such as ribo-somes, but not the smaller tRNA molecules. He used anylon filter to separate ribosomes (and anything bound tothe ribosomes) from unbound tRNA. To detect the tRNAbound to the ribosomes, Nirenberg used tRNA chargedwith amino acids that contained a radioactive label, C.All other components of the reaction were not radioactive.Since only ribosome-bound tRNA is retained by the nylon

14

membrane, all radioactivity found on the nylon membranecorresponds to tRNA bound to ribosomes. Now, a systemwas in place to detect the recognition between a mRNAmolecule and the proper amino-acylated tRNA.

To test his system, Nirenberg used polyU as themRNA, and [ C]-phenylalanine-charged tRNA whichbinds to ribosomes in the presence of polyU. Ribosomeswere incubated with both polyU and [ C]-phenylalaninetRNA for sufficient time to allow both molecules to bindto the ribosomes; the reaction mixtures were then passedthrough a nylon membrane. When the membranes wereanalyzed using a scintillation counter, they containedradioactivity, demonstrating that in this system polyUcould recognize phenylalanine-charged tRNA. But wasthis recognition specific for the proper amino-acylatedtRNA? As a control, [ C]-lysine and [ C]-proline-charged tRNAs also were incubated with polyU and ribo-somes. After the reaction mixtures were passed through anylon filter, no radioactivity was detected on the filter.Therefore, the assay measured only specific bindingbetween a mRNA and its corresponding amino-acylatedtRNA.

Now, the minimum chain length of RNA necessary forproper amino-acylated tRNA recognition could be deter-mined. Short oligonucleotides were tested for their abilityto bind ribosomes and recognize the appropriate tRNA.

1414

14

14

PheTrinucleotide and all tRNAspass through filter

Trinucleotide

Aminoacyl-tRNAs

Ribosomes stick to filter

Ribosomes

Complex of ribosome, UUU,and Phe-tRNA sticks to filter

UUU

UUU

UUU

UUU

Phe

Phe

Leu

Leu

Leu

Arg Arg

Arg

Arg

Phe

Leu

Assay developed by Marshall Nirenberg and his collaborators for deciphering the genetic code. They prepared 20 E. coli extracts contain-ing all the aminoacyl-tRNAs (tRNAs with amino acid attached). In each extract sample, a different amino acid was radioactively labeled(green); the other 19 amino acids were present on tRNAs but remained unlabeled. Aminoacyl-tRNAs and trinucleotides passed through anylon filter without binding (left panel); ribosomes, however, bind to the filter (center panel). Each of the 64 possible trinucleotides wastested separately for its ability to attract a specific tRNA by adding it with ribosomes to different extract samples. Each sample was thenfiltered. If the added trinucleotide causes the radiolabeled aminoacyl-tRNA to bind to the ribosome, then radioactivity is detected on thefilter; otherwise, the label passes through the filter (right panel). By synthesizing and testing all possible trinucleotides, the researcherswere able to match all 20 amino acids with one or more codons (e.g., phenyalanine with UUU as shown here). [From H. Lodish et al.,1995, Molecular Cell Biology, 3rd ed. W. H. Freeman and Company. See M. W. Nirenberg and P. Leder, 1964, Science 145:1399].


When UUU, a trinucleotide, was used, tRNA binding toribosomes could be detected. However, when the UU di-nucleotides was used, no binding was detected. This resultsuggested that the codon required for proper recognitionof tRNA is a trinucleotide. Nirenberg repeated this exper-iment on two other homogeneous trinucleotides, CCC andAAA. When these trinucleotides were independentlybound to ribosomes, CCC specifically recognized prolinecharged tRNA and AAA recognized lysine-chargedtRNAs. Since none of the three homogeneous trinu-cleotides recognized other charged tRNAs, Nirenberg con-cluded that trinucleotides could effectively direct the properrecognition of amino-acylated tRNAs.

This study accomplished much more than determiningthe length of the codon required for proper tRNA recogni-tion. Nirenberg realized that his assay could be used to testall 64 possible combinations of trinucleotides (see Figure).A method for cracking the code was available!

DiscussionCombined with the technology to generate trinucleotidesof known sequence, Nirenberg’s assay provided a way toassign each specific amino acid to one or more specifictrinucleotides. Within a few years, the genetic code wascracked, all 20 amino acids were assigned at least one tri-nucleotide, and 61 of the 64 trinucleotides were found tocorrespond to an amino acid. The final three trinu-cleotides, now known as “stop” codons, signal termina-tion of protein synthesis.

With the genetic code cracked, biologists could readthe gene in the same manner that the cell did. Simply byknowing the DNA sequence of a gene, scientists can nowpredict the amino acid sequence of the protein it encodes.For his innovative work, Nirenberg was awarded theNobel Prize for Physiology and Medicine in 1968.


BackgroundIn 1961, Howard Temin began to gather evidence thatwas inconsistant with the central dogma. Temin, whodevoted his life to studying RNA tumor viruses (nowknown as retroviruses), focused his early work on Roussarcoma virus (RSV). This RNA virus is capable of trans-forming normal cells into cancerous cells. Temin felt thebest explanation for the virus’s behavior was a modelwhereby the virus remains in a dormant, or proviral, statein the cell. However, since RNA is notoriously unstable,Temin proposed that the RNA genome of RSV is convertedinto a DNA provirus. With this model in mind, he set outto prove his hypothesis. He amassed data showing thatRSV is sensitive to inhibitors of DNA synthesis and sug-gesting that DNA, complementary to the RSV genomicRNA, is present in transformed cells. Other researchers,however, remained unconvinced. A definitive experimentwould be required to finally prove his model.

Meanwhile, another virologist, David Baltimore, hadbeen studying the replication of viruses. He was taking abiochemical approach, looking directly for RNA andDNA synthesis in the virions themselves. Previously hehad isolated an RNA-dependent RNA polymerase activityin virions of vesicular stomatitis virus, a nontumorigenicRNA virus. His attention then turned to the RNA tumorviruses, finally settling on the Rauscher murine leukemia

virus (R-MLV). With this organism, he would indepen-dently prove Temin’s model.

The ExperimentRemarkably, these two scientists traveled separate path-ways to the same critical set of experiments. Both beganwith pure stocks of virus, which they then partially dis-rupted using nonionic detergents. With a stock of disrupted viruses in hand they could ask the critical ques-tion: Can a retrovirus perform DNA synthesis? To answerthis question, each groups added radiolabeleddeoxythymidine triphosphate (dTTP) along with the otherthree deoxynucleotide triphosphates (dATP, dCTP, dGTP)to the virion preparations, and looked for the incorpora-tion of radioactive dTTP into DNA. Indeed, in each exper-iment radiolabeled dTTP was incorporated into nucleicacid. When Baltimore added a radiolabeled ribonucleotidetriphosphate (rNTP) and the three other ribonucleotidetriphosphates to disrupted viruses, he could detect no RNAsynthesis. To prove that the product being formed was infact DNA, they treated it with enzymes that specificallydegrade either RNA (ribonuclease, or RNase) or DNA(deoxyribonuclease, or DNase). They found the product tobe sensitive only to DNase. The results of these simpleexperiments, summarized in the Table, showed that an

THE DISCOVERY OF REVERSETRANSCRIPTASE

Through a series of experiments conducted in the 1940s, 1950s, and 1960s,

the principles by which genetic information is transferred in biological sys-

tems were demonstrated. DNA served as the code, which was then transcribed

into a type of RNA (mRNA), that carried the message to be translated into pro-

teins. These experiments formed a paradigm so firmly believed it was known as

the “central dogma.” However, in 1970, work on RNA tumor viruses showed

that perhaps the central dogma did not explain the whole picture.



enzyme in the particles could synthesize DNA. However,the question remained . . . What was the template?

To show once and for all that DNA could be synthe-sized from an RNA template, Baltimore and Temin bothpreincubated the virions with RNase, which catalyzes thedegradation of RNA into ribonucleotide monophosphates(rNMPs). If RNA was truly the template, then degredationof the template would prevent DNA synthesis by the viri-on preparations. This in fact was the case. The longer thepretreatment of virions with RNase, the lower the amountof DNA-synthesizing activity, thus proving that an enzymein the virion could catalyze RNA-dependent DNA synthe-sis. Because this activity was the reverse of the well-knownDNA-dependant RNA synthesis seen in transcription, theenzyme that catalyzed it was soon refereed to as reversetranscriptase. Intially, many scientists were unwilling tobelieve that reverse transcriptase existed because its activ-ity violated the central dogma. Subsequent isolation andcharacterization of the paradigm-shattering enzyme soonconvinced the skeptics.

DiscussionTemin and Baltimore were led independently by differentkey deductions to the discovery of reverse transcriptase.

Temin firmly believed the activity existed. For him, it wasthe process of doing biochemical experiments on purifiedvirions, rather than on infected cells, that allowed him to prove to the world what he knew. Baltimore, on the otherhand, believed that viruses carried their polymerase activi-ties with them. His key insight was to test for the RNA-dependent DNA polymerization activity that Temin hadproposed. Both scientists, however, had to have the convic-tion to believe and report what they were seeing, despite itsbeing contrary to a seemingly unshakable paradigm.

The discovery of reverse transcriptase has impacted lifein and out of science in a myriad of ways. The ability toconvert mRNA to DNA permitted creation of cDNAlibraries, collections of DNA made up solely of genesexpressed in a particular tissue. This has facilitated thecloning and study of genes involved in all facets of biol-ogy. The discovery also caused an explosion of researchinto retroviruses, RNA viruses that replicate via reversetranscription. This groundwork was critical 15 years laterwhen the human innunodeficiency virus (HIV), whichcauses AIDS, was shown to be a retrovirus. The impor-tance of Temin’s and Baltimore’s work was quickly recog-nized, leading to their receiving the Nobel Prize forPhysiology and Medicine in 1975 for the discovery ofreverse transcriptase.

Demonstration of RNA-dependent DNA Synthesis

Radioactive Thymidine Incorporated into Nucleic Acid*

Experimental treatment R-MLV RSV

Standard conditions 3.69 pmol 9110 dpmVirions pretreated with RNase 0.52 pmol 2650 dpm

Untreated product 1425 dpm 8350 dpmAfter product is treated with RNase 1361 dpm 7200 dpmAfter product is treated with DNase 125 dpm 1520 dpm

*dpm disintegrations per minute; pmols picmoles.SOURCE: R-MLV data from D. Baltimore, 1970, Nature 226:1209. RSV data from H. M. Temin and S. Mizutani, 1970, Nature 226:1211.

��


BackgroundDuring the 1950s, scientists uncovered many of biologicalfacts we now take for granted, beginning with the discov-ery that genetic information is passed on through deoxyri-bonucleic acid (DNA), and continuing through the eluci-dation of DNA’s three-dimensional structure. As thedecade neared a close, biologists were ready to study howDNA passed on genetic information from the parental tothe progeny generation.

James Watson and Francis Crick had hypothesized,based on their double-helical model of DNA, that replica-tion occurs in a semiconservative fashion. That is, thedouble helix unwinds, the original parental DNA standsserve as templates to direct the synthesis of the progenystrand, and each of the replicated DNA duplexes containsone old (parental) strand, and one newly synthesizedstrand, often called the “daughter” strand. Anotherhypothesis proposed at the time was conservative replica-tion, whereby after replication the parental strandsformed one DNA duplex and the two daughter standsformed the second duplex.

When these hypotheses were first proposed, littleexperimental evidence was available to support one overanother. In 1957, however, Messelson and Stahl, alongwith Jerome Vinograd, developed density-gradient cen-trifugation, a technique that can separate macromolecules

exhibiting very small differences in density. The tools werenow available for a definitive test to determine whetherDNA replication occurs by a semiconservative or conserv-ative mechanism.

The ExperimentMeselson and Stahl reasoned that if one could label theparental DNA in such a way that it could be distinguishedfrom the daughter DNA, the replication mechanisms couldbe distinguished. If DNA replication is semiconservative,then after a single round of replication, all DNA moleculesshould be hybrids of parental and daughter DNA strands.If replication is conservative, then after a single round ofreplication, half of the DNA molecules should be composedonly of parental strands and half of daughter strands.

To differentiate parental DNA from daughter DNA,Messelson and Stahl used “heavy” nitrogen (15N). Thisisotope contains an extra neutron in its nucleus, giving ita higher atomic mass than the more abundant “light”nitrogen (14N). Since nitrogen atoms make up part of thepurine and pyrimidine bases in DNA, it was easy to labelE. coli DNA with 15N by growing bacteria in a mediumcontaining 15N ammonium salts as the sole nitrogensource. After several generations of growth, the bacteriacontained only 15N-labeled DNA. Now that the parental

PROVING THAT DNA REPLICATION ISSEMICONSERVATIVE

The discovery that the structure of DNA is a double helix, containing two

complementary strands of DNA, led to a number of hypotheses about how

DNA might be replicated. Although the possible replication mechanisms were rel-

atively easy to deduce, proving which occurs in vivo was a more difficult task. In

1958, Mathew Meselson and Franklin Stahl used the newly developed techniques

of density-gradient centrifugation, to show that DNA replication proceeds in a

semiconservative fashion.



DNA was labeled, Meselson and Stahl abruptly changedthe medium to one containing 14N as the sole nitrogensource. From this point on, all the DNA synthesized by thebacteria would incorporate 14N, rather than 15N, so thatthe daughter DNA strands would contain only 14N. As thebacteria continued to grow and replicate their DNA in the14N-containing medium, samples were taken periodicallyand the bacterial DNA was analyzed with the newly devel-oped technique of equilibrium density-gradient centrifuga-tion. In this type of analysis, a DNA sample is mixed witha solution of cesium chloride (CsCl2). During long periodsof high-speed centrifugation the CsCl2 forms a gradient,and the DNA migrates to the position where the density ofthe DNA is equal to that of the CsCl2. If the DNA samplecontains molecules of different densities they will migrateto different positions in the gradient. Because 15N has agreater density than 14N, 15N-labeled DNA has a greaterdensity than 14N-labeled DNA. The higher-density (15N)DNA will sediment to a different position than the lower-density (14N) DNA. Hybrid DNA molecules, containingboth 15N and 14N, will sediment at an intermediate den-sity, depending on the ratio of heavy nitrogen to lightnitrogen.

The Figure illustrates the results obtained by Meselsonand Stahl. Before any DNA replication had occurred in the14N-containing medium, all DNA sedimented as a singlespecies, corresponding to 15N-labeled DNA. As DNA repli-cation proceeded, the amount of (15N)-DNA decreased, anda second DNA species, consisting of hybrid DNA moleculescontaining 15N- and 14N-labeled strands, appeared. DNAcollected after completion of the first round of replicationwas found to sediment with the second species. When the

DNA produced during a second round of replication wasanalyzed, two distinct species were observed. One corre-sponded to hybrid molecules; the other corresponded to14N-labeled DNA. With each subsequent round of replica-tion the proportion of hybrid DNA decreased as theamount of 14N-labeled DNA increased. As the diagrams inthe Figure show, the sedimentation patterns observed byMesselson and Stahl are consistent only with a semiconser-vative model of replication.

DiscussionFor Meselson and Stahl to prove that DNA replicationproceeds in a semiconservative manner, they not onlyhad to design a clear, easily interpretable experiment,but also develop the technology to do it. The beauty ofthis classic experiment is that each of the possible mod-els would produce distinctly different results, so thatinterpretation of the experimental data was unambigu-ous. This study remains a shining example of defining aproblem and employing the proper methodology tosolve it.

By demonstrating that DNA replication occurs in asemi-conservative fashion, Meselson and Stahl opened upthe field of DNA replication for in depth research. Withthe correct model in hand, researchers could now turn tounraveling the precise mechanism of DNA replication. Inaddition, equilibrium density-gradient centrifugationbecame a widely used tool for the analysis of complexmixtures of DNA.


Oldstrand

Parent strandssynthesizedin 15N

First doublingin 14N

Second doublingin 14N

One band:H–H

Two bands:H–H + L–L

New strands

One bandH–H

One band:H–L (hybrid)

Two bands:H–L + L–L

Two bands:H–H + L–L

Light(14N)

Heavy(15N)

H–HH H

L L L H H LHH

H H L L L L L L H L L L H L L L

H H

Newstrand

+ +

Conservative model

Predicted results Actual results

Semiconservative model

H–L

L–L H–L

Experimental demonstration by Messelson and Stahl that DNA replication is semiconservative. After several generations of growth in amedium containing “heavy” (15N) nitrogen, E. coli were transferred to a medium containing the normal “light” isotope (14N). Sampleswere removed from the cultures periodically and analyzed by equilibrium density-gradient centrifugation in CsCl to separate heavy-heavy(H-H), light-light (L-L), and heavy-light (H-L) duplexes into distinct bands. The actual banding patterns observed were consistent with thesemiconservative mechanism. [From H. Lodish et al., 1995, Molecular Cell Biology, 3rd ed. W. H. Freeman and Company. See M.Messelson and W. F. Stahl, 1958, Proc. Nat’l. Acad. Sci. USA 44:671; photographs courtsey of M. Messelson.]


BackgroundEukaryotic cells are highly organized and composed of cellstructures known as organelles that perform specific func-tions. While microscopy has allowed biologists to describethe location and appearance of various organelles, it is oflimited use in uncovering the organelle’s function. To dothis, cell biologists have relied on a technique known ascell fractionation. Here, cells are broken open, and the cel-lular components are separated on the basis of size, mass,and density using a variety of centrifugation techniques.Scientists could then isolate and analyze cell componentsof different densities, called fractions. Using this method,biologists had divided the cell into four fractions: nuclei,mitochondrial-rich fraction, microcosms, and cell sap.

de Duve was a biochemist interested in the subcellularlocations of metabolic enzymes. He had already completeda large body of work on the fractionation of liver cells, inwhich he had determined the subcellular location of nu-merous enzymes. By locating these enzymes in specific cellfractions, he could begin to elucidate the function of theorganelle. He has noted that his work was guided by twohypotheses: the “postulate of biochemical homogeneity”and “the postulate of single location.” In short, these hy-potheses propose that the entire composition of a subcel-lular population will contain the same enzymes, and that

SEPARATING ORGANELLES

In the 1950s and 1960s, scientists used two techniques to study cell organelles:

microscopy and fractionation. Christian de Duve was at the forefront of cell

fractionation. In the early 1950s, he used centrifugation to distinguish a new

organelle, the lysosome, from previously characterized fractions: the nucleus,

the mitochondrial-rich fraction, and the microsomes. Soon thereafter, he used

equilibrium-density centrifugation to uncover yet another organelle.


each enzyme is located at a discrete site within the cell.Armed with these hypotheses and the powerful tool of cen-trifugation, de Duve further subdivided the mitochondrial-rich fraction. First, he identified the light mitochondrialfraction, which is made up of hydrolytic enzymes that arenow known to compose the lysosome. Then, in a series ofexperiments described here, he identified another discretesubcellular fraction, which he called the perioxisome,within the mitochondrial-rich fraction.

The Experimentde Duve studied the distribution of enzymes in rat livercells. Highly active in energy metabolism, the liver con-tains a number of useful enzymes to study. To look for thepresence of various enzymes during the fractionation, herelied on known tests, called enzyme assays, for enzymeactivity. To retain maximum enzyme activity, he had totake precautions, which included performing all fraction-ation steps at 0°C because heat denatures protein thatwould compromise enzyme activity.

de Duve used rate-zonal centrifugation to separate cel-lular components by successive centrifugation steps. Heremoved the rat’s liver and broke it apart by homoge-nization. The crude preparation of homogenized cells was


then subjected to relatively low-speed centrifugation. Thisinitial step separated the cell nucleus, which collects assediment at the bottom of the tube, from the cytoplasmicextract that remains in the supernatant. Next, de Duvefurther subdivided the cytoplasmic extract into heavy mi-tochondrial fraction, light mitochondrial fraction, and mi-crosomal fraction. He accomplished separating the cyto-plasm by employing successive centrifugation steps ofincreasing force. At each step, he collected and stored thefractions for subsequent enzyme analysis.

Once the fractionation was complete, de Duve per-formed enzyme assays to determine the subcellular distri-bution of each enzyme. He then graphically plotted thedistribution of the enzyme throughout the cell. As hadbeen shown previously, the activity of cytochrome oxidase,an important enzyme in the electron transfer system, wasfound primarily in the heavy mitochondrial fractions. Themicrosomal fraction was shown to contain another previ-ously characterized enzyme glucose-6-phosphatase. Thelight mitochondrial fraction, which is made up of the lyso-some, showed the characteristic acid phosphatase activity.Unexpectedly, de Duve observed a fourth pattern when heassayed the uricase activity. Rather than following the pat-tern of the reference enzymes, uricase activity was sharplyconcentrated within the light mitochondrial fraction. Thissharp concentration, in contrast to the broad distribution,suggested to de Duve that the uricase might be secludedin another subcellular population separate from the lyso-somal enzymes.

To test this theory, de Duve employed a techniqueknown as equilibrium density-gradient centrifugation,

which separates macromolecules on the basis of density.Equilibrium density-gradient centrifugation can be per-formed using a number of different gradients including su-crose and glycogen. In addition, the gradient can be madeup in either water or “heavy water” that contains the hy-drogen isotope deuterium in place of hydrogen. In his ex-periment, de Duve separated the mitochondrial-rich frac-tion prepared by rate-zonal centrifugation in each of thesedifferent gradients (see Figure 5.1). If uricase were part ofa separate subcellular compartment, it would separatefrom the lysosomal enzymes in each gradient tested. deDuve performed the fractionations in this series of gradi-ents, then performed enzyme assays as before. In each case,he found uricase in a separate population than the lyso-somal enzyme acid phosphatase and the mitochondrial

Organellefraction

Lysosomes(1.12 g/cm3)

Mitochondria(1.18 g/cm3)

Peroxisomes(1.23 g/cm3)

Beforecentrifugation

Aftercentrifugation

Incr

easi

ng

den

sity

of

sucr

ose

(g

/cm

3 )

1.09

1.11

1.15

1.19

1.22

1.25

5

4

20 40 60 80

3

1

2

5

4

20 40 60 80

3

1

2

Rel

ativ

e co

nce

ntr

atio

n

Cytochrome oxidase

Uricase

Acid phosphatase

5

4

20 40 60 80

3

1

2

Percent height in tube

▲ FIGURE 5.1 Schematic depiction of the separation of the

lysosomes, mitochondria, and perioxisomes by equilibrium

density centrifugation. The mitochondrial-rich fraction from rate-zonal centrifugation was separated in a sucrose gradient, and theorganelles are separated on the basis of density. [From Lodish et al., 3rd edition, page 166.]

▲ FIGURE 5.2 Graphical representation of the enzyme

analysis of products from a sucrose gradient. The mitochon-drial-rich fraction was separated as depicted in Figure 5.1, andthen enzyme assays were performed. The relative concentrationof active enzyme is plotted on the y-axis; the height in the tube isplotted on the x-axis. The peak activities of cytochrome oxidase(top) and acid phosphatase (bottom) are observed near the top oftube. The peak activity of uricase (middle) migrates to the bottomof the tube. [Adapted from Beaufay et al., 1964, Biochem J.92:191.]


enzyme cytochrome oxidase (see Figure 5.2). By repeat-edly observing uricase activity in a distinct fraction fromthe activity of the lysosomal and mitochondrial enzymes,de Duve concluded that uricase was part of a separate or-ganelle. The experiment also showed that two other en-zymes, catalase and D-amino acid oxidase, segregated intothe same fractions as uricase. Because each of these en-zymes either produced or used hydrogen peroxide, deDuve proposed that this fraction represented an organelleresponsible for the peroxide metabolism and dubbed it theperioxisome.

Discussionde Duve’s work on cellular fractionation provided an in-sight into the function of cell structures, as he sought to

map the location of known enzymes. Examining the inven-tory of enzymes in a given cell fraction gave him clues toits function. His careful work resulted in the uncovering oftwo organelles: the lysosome and the perioxisome. His workalso provided important clues to the organelles’ function.The lysosome, where de Duve found so many potentiallydestructive enzymes, is now known to be an important sitefor degradation of biomolecules. The perioxisome has beenshown to be the site of fatty acid and amino acid oxida-tion, reactions that produce a large amount of hydrogenperoxide. In 1974, de Duve received the Nobel Prize forPhysiology and Medicine in recognition of his pioneeringwork.


BRINGING CELLS TOGETHER

The surfaces of many animal cells are coated with cell adhesion molecules

including integral membrane proteins that mediate the cell-cell interactions

critical for tissue formation. In the late 1970s and early 1980s, biologists began to

identify some of these molecules. During this time, the Japanese scientist

Masatoshi Takeichi showed that some molecules mediate cell-cell interactions

only in the presence of Ca2� ions. This observation led to the discovery of a new

class of adhesion molecules, the cadherins.


conditions under which they would and would not adhereto one another. At the same time, other researchers beganto identify specific molecules that mediate cell adhesiveinteractions. Taken together, these two approaches wouldlead to the discovery of cadherins, a group of cell adhesivemolecules critical for tissue formation during development.

The ExperimentIn the late 1970s, when Takeichi studied the adhesive prop-erties of a lung cell line in culture, he observed that calci-um was critical for some forms of cell adhesion. Similar toother cultured cells, the lung cells would readily dissociatein the presence of the protease trypsin. The dissociated cellswould normally reaggregate when the trypsin was washedaway. However, when Takeichi attempted to replicate theseresults in a different laboratory, he found that the cellsremained dissociated after trypsin treatment, and once dis-sociated, the cells would never aggregate again under thenew conditions.

Puzzled by his difficulty in repeating this seeminglybasic experimental procedure, Takeichi looked at thechemical compositions of the solutions used in his newlaboratory. He found that the trypsin solution he used inthe new laboratory contained EDTA, a chemical thatsequesters divalent cations from the solution and thus

BackgroundIn multicellular organisms, groups of specialized cells cometogether to form tissues. This grouping of cells is not ran-dom; specific cell types must adhere to one another. Specificinteractions between cells assure that cellular composition iscorrect: epithelial cells are found in epithelium, hepatocytesin the liver, and neurons in neuronal tissues such as thebrain. During tissue formation, cells of the same type inter-act with one another and avoid interactions with other celltypes. In organs, where cells of many types work together,the interaction between different cell types is specific.Clearly, there must be a mechanism to assure that tissuesand organs maintain the correct cellular compositions.

Many researchers study the adhesive interactions thatoccur in tissues using embryonic cells in culture. Thesecells will adhere to one another in interactions so tightlythat a protease must be added to break them apart. Classicexperiments performed in the 1960s showed that whendifferent cell types are placed in the same Petri dish, theyseparate from each other like oil and water. Thus, in cellculture, just as in the body, cells adhere to cells of the sametype and avoid contact with different types of cells. Butthe question remained, how are these specific adhesiveinteractions achieved?

In the late 1970s, Masatoshi Takeichi studied the inter-actions between cells in cultures and attempted to find the

8945d_001-004 3/11/05 10:23 PM Page 1 EQA

body is found, researchers use it to identify a specific pro-tein involved in the cell adhesion. At first, Takeichi usedthe same lung cell line that he used to demonstrate thatsome cell adhesions are Ca2�-dependent. However, hewas unable to find an antibody that would block celladhesion in this cell line.

To overcome this problem, Takeichi began studyingcell adhesion in a different cell line, a teratocarcinoma cellline, where Ca2�-dependent cell adhesion also occurs. Hegrew cells in the presence of Ca2�, used trypsin to dissoci-ate them, and then injected them into rabbits, whoseimmune system generated antibodies that recognize pro-teins on the surface of these cells. To purify these antibod-ies, he treated the teratocarcinoma cells that had not beenexposed to calcium with antiserum taken from the inject-ed rabbits. This treatment removed all the antibodies thatbound to teratocarcinoma cells in both the presence andthe absence of Ca2�. What remained were antibodies thatspecifically bind proteins that are on the cell surface in thepresence of Ca2�.

Fibroblasts expressing E-cadherin adhere in culture. Cells in (a) and (c) are from a fibroblast cell line growing in culture. Cells in(b) and (d) are fibroblasts from the same cell line transfected with the cDNA encoding E-cadherin. (a) Light micrograph showing thatfibroblasts do not form adhesive interactions in culture. Notice how the cells seem to overlap one another. (b) Light micrograph showingfibroblasts expressing E-cadherin in culture. These cells adhere to one another, as demonstrated by the easily definable boundariesbetween cells. (c) Immunofluorescence experiment showing that fibroblasts in culture do not normally express E-cadherin. (d)Immunofluorescence staining shows that fibroblasts transfected with the cDNA encoding E-cadherin express the molecule on their cellsurfaces, suggesting that E-cadherin is in fact mediating cell adhesion. (Nagafuchi, A., et al. [1987]. Nature 329: 341–343.)

from the lung cells. Previously, Takeichi had used a solu-tion that did not contain EDTA. Perhaps a divalent cationwas involved in the adhesive interactions between thesecells? To find out, Takeichi began investigating the effectsof divalent cations on the adhesive properties of the lungcells. He found that the cells would not dissociate in thepresence of Ca2� and that dissociated cells would reasso-ciate only when Ca2� was added to the medium. Theseobservations led him to propose that some types of celladhesions depend on calcium.

Next, Takeichi set out to identify the specific mole-cules involved in Ca2�-dependent cell adhesion. He usedantibodies raised against cell surface proteins involved incell-cell adhesions to identify the specific proteins, a strat-egy similar to that used to identify other cell adhesionproteins. The basis of this strategy is the observation thatwhen cultured cells are treated with these antibodies, thebinding sites of adhesion molecules are blocked and theinteractions between them cannot take place. As a result,the cells no longer adhere in culture. Once such an anti-

(a) (b)

(d)(c)

8945d_001-004 3/23/05 5:53 PM Page 2 EQA

fibroblasts and thus were probably responsible for theirnewly acquired adhesive property (Figures c and d).

DiscussionOver a 10-year period, Takeichi and colleagues used hisobservation about the dependency of some adhesive inter-actions on the divalent cation Ca2� to discover a key classof cell surface molecules and to show that they are criticalfor Ca2�-dependent cell adhesions. The discovery of Ca2�-dependent cell adhesion was prompted when an experi-ment that had always worked, the reassociation of cellsthat had been dissociated by trypsin, suddenly stoppedworking in the conditions of the new laboratory. By track-ing down the difference between the conditions of the lab-oratories, Takeichi made the initial observations that led tothe discovery of a critical family of cell adhesion molecules.His work shows that sometimes great science comes fromwhat looks like a failed experiment.

Today, different cadherins have been identified on var-ious types of tissues from the placenta to neurons. Laterexperiments would show that each type of cadherin inter-acts specifically with the same molecule on an adjacentcell; in other words, an E-cadherin interacts with anotherE-cadherin but not with an N-cadherin. These homophilicinteractions provide some of the specificity that allows tis-sues to form. The discovery of cadherins led the way toour understanding of how tissues form.

To find the molecule involved in Ca2�-dependent celladhesion, Takeichi compared cultures grown in the pres-ence of Ca2� with cultures of cells grown in the presenceof EDTA, which sequesters Ca2� ions. Both groups ofcells were dissociated with the protease trypsin before theexperiment began. Using a technique called immunopre-cipitation, he showed that his antibody specifically inter-acted with a 140 kDa protein on the surface of the Ca2�-treated cells while it did not specifically interact with anyprotein on the EDTA-treated cells. He named this proteincadherin for calcium-dependent adhesion protein. Asmore cadherins were discovered, the protein becameknown as E-cadherin because it mediates the adhesion ofepithelial cells.

Takeichi’s identification of E-cadherin demonstrated thatthe protein is involved in Ca2�-mediated cell adhesion, buthis research did not prove that E-cadherin is primarilyresponsible for this type of adhesion. To do so, Takeichi andcolleagues cloned the gene encoding E-cadherin. Once thegene was cloned, he could express E-cadherin in a fibroblastcell line that neither expressed E-cadherin nor demonstratedCa2�-dependent adhesion. Rather than form cell-cell adhe-sions, fibroblasts grow on top of one another in culture(Figure a). However, fibroblasts that express E-cadherinadhered to one another in the presence of Ca2� (Figure b).Thus, the expression of one protein, E-cadherin, changed theadhesive properties of the fibroblast cell line. Finally,Takeichi used immunofluorescence microscopy to show thatthe E-cadherin molecules are found at the cell surface of the

8945d_001-004 3/23/05 5:53 PM Page 3 EQA

8945d_001-004 3/11/05 10:23 PM Page 4 EQA

BackgroundDuring the 1950s many researchers around the worldwere actively investigating the physiology of the cell mem-brane, which plays a role in a number of biologicalprocesses. It was well known that the concentration ofmany ions differs inside and outside the cell. For example,the cell maintains a lower intracellular sodium (Na�) con-centration and higher intracellular potassium (K�) con-centration than is found outside the cell. Somehow themembrane can regulate intracellular salt concentrations.Additionally, movement of ions across cell membraneshad been observed, suggesting that some sort of transportis system is present. To maintain normal intracellular Na�

and K� concentrations, the transport system could notrely on passive diffusion because both ions must moveacross the membrane against their concentration gradients.This energy-requiring process was termed active transport.

At the time of Skou’s experiments, the mechanism ofactive transport was still unclear. Surprisingly, Skou hadno intention of helping to clarify the field. He found theNa�/K� ATPase completely by accident in his search foran abundant, easily measured enzyme activity associatedwith lipid membranes. A recent study had shown thatmembranes derived from squid axons contained a mem-brane-associated enzyme that could hydrolyze ATP.Thinking that this would be an ideal enzyme for his pur-

STUMBLING UPON ACTIVE TRANSPORT

Bn the mid-1950s Jens Skou was a young physician researching the effects of

local anesthetics on isolated lipid bilayers. He needed an easily assayed mem-

brane-associated enzyme to use as a marker in his studies. What he discovered

was an enzyme critical to the maintenance of membrane potential, the Na1/K1

ATPase, a molecular pump that catalyzes active transport.


poses, Skou set out to isolate such an ATPase from a morereadily available source, crab leg neurons. It was duringhis characterization of this enzyme that he discovered theprotein’s function.

The ExperimentSince the original goal of his study was to characterize theATPase for use in subsequent studies, Skou wanted toknow under what experimental condition its activity wasboth robust and reproducible. As often is the case with thecharacterization of a new enzyme, this requires carefultitration of the various components of the reaction. Beforethis can be done, one must be sure the system is free fromoutside sources of contamination.

In order to study the influence of various cations,including three that are critical for the reaction—Na�,K�, and Mg2�—Skou had to make sure that no contami-nating ions were brought into the reaction from anothersource. Therefore, all buffers used in the purification ofthe enzyme were prepared from salts that did not containthese cations. An additional source of contaminatingcations was the ATP substrate, which contains three phos-phate groups, giving it an overall negative charge. Becausestock solutions of ATP often included a cation to balancethe charge, Skou converted the ATP used in his reactions


to the acid form, so that balancing cations would notaffect the experiments. Once he had a well-controlledenvironment, he could characterize the enzyme activity.These precautions were fundamental to his discovery.

Skou first showed that his enzyme could indeed cat-alyze the cleavage of ATP into ADP and inorganic phos-phate. He then moved on to look for the optimal condi-tions for this activity by varying the pH of the reaction,and the concentrations of salts and other cofactors, whichbring cations into the reaction. He could easily determinea pH optimum as well as an optimal concentration ofMg2�, but optimizing Na� and K� proved to be moredifficult. Regardless of the amount of K� added to thereaction, the enzyme was inactive without Na�. Similarly,without K�, Skou observed only a low-level ATPaseactivity that did not increase with increasing amounts ofNa�.

These results suggested that the enzyme required bothNa� and K� for optimal activity. To demonstrate that thiswas the case, Skou performed a series of experiments inwhich he measured the enzyme activity as he varied boththe Na� and K� concentrations in the reaction (seeFigure). Although both cations clearly were required forsignificant activity, something interesting occurred at highconcentrations of each cation. At the optimal concentra-tion of Na� and K�, the ATPase activity reached a peak.Once at that peak, further increasing the concentrationdid not affect the ATPase activity. Na� thus behaved like

a classic enzyme substrate, with increasing input leadingto increased activity until a saturating concentration wasachieved, at which the activity plateaued. K�, on the otherhand, behaved differently. When the K� concentrationwas increased beyond the optimum, ATPase activitydeclined. Thus, while K� was required for optimal activi-ty, at high concentrations it inhibited the enzyme. Skoureasoned that the enzyme must have separate binding sitesfor Na� and K�. For optimal ATPase activity, both mustbe filled. However, at high concentrations K� could com-pete for the Na�-binding site, leading to enzyme inhibi-tion. He hypothesized that this enzyme was involved inactive transport, that is, the pumping of Na� out of thecell, coupled to the import of K� into the cell. Later stud-ies would prove that this enzyme was indeed the pumpthat catalyzed active transport. This finding was so excit-ing that Skou devoted his subsequent research to studyingthe enzyme, never using it as a marker, as he initiallyintended.

DiscussionSkou’s finding that a membrane ATPase used both Na�

and K� as substrates was the first step in understandingactive transport on a molecular level. How did Skou knowto test both Na� and K�? In his Nobel lecture in 1997, heexplained that in his first attempts at characterizing the

(a)

0

(b)

µgP

40

30

20

10

0

µgP

40

30

20

10

020 40 8060 100 120

KCl mM/ I

0 10050 150 200

NaCl mM/ I

K 0 mM/ I

Mg 6 mM/ I

Mg 6 mM/ I

NaCl 40 mM/ I

NaCl 20 mM/ I

NaCl 10 mM/ I

NaCl 0 mM/ I

NaCl 3 mM/ I

K 350 mM/ I

K 200 mM/ I

K 120 mM/ IK 20 mM/ I

K 3 mM/ I

Demonstration of the dependence of the Na�/K� ATPase activity on the concentration of each ion. The graph on the left shows thatincreasing K� leads to an inhibition of the ATPase activity. The graph on the right shows that with increasing Na�, the enzyme activityincreases up to a peak and then levels out. This graph also demonstrates the dependence of the activity on low levels of K�. [Adaptedfrom J. Skou, 1957, Biochem. Biophys. Acta 23:394.]


ATPase, he took no precautions to avoid the use of buffersand ATP stock solutions that contained Na� and K�.Pondering the puzzling and unreproducible results that heobtained led to the realization that contaminating saltsmight be influencing the reaction. When he repeated theexperiments, this time avoiding contamination by Na�

and K� at all stages, he obtained clear-cut reproducibleresults.

The discovery of the Na�/K� ATPase had an enor-mous impact on membrane biology, leading to a betterunderstanding of the membrane potential. The generationand disruption of membrane potential forms the basis ofmany biological processes including neurotransmissionand the coupling of chemical and electrical energy. For thisfundamental discovery, Skou was awarded the NobelPrize for Chemistry in 1997.


BackgroundResearchers in the human genetics department of a youngbiotechnology company were trying to develop a practicalmethod for the prenatal diagnosis of sickle cell anemia.The molecular defect that causes most cases of this diseaseis a single nucleotide change in the sixth codon of the geneencoding the protein �-globin, one of the subunits ofhemoglobin. Kary Mullis, a molecular biologist at thecompany, had an idea for a molecular method that wouldamplifiy specific DNA sequences. The detection of a singlenucleotide change, as occurs in sickle cell anemia, was theperfect test for his ideas.

Mullis’s idea was an extension of known techniquesfor synthesizing specific pieces of DNA in vitro usingchemically synthesized oligonucleotides and purified DNApolymerase, the enzyme that catalyzes the synthesis ofDNA. First, a short oligonucleotide whose sequence wascomplementary to a portion of the target DNA was syn-thesized. Next, a fragment of DNA containing the targetsequence was isolated using restriction endonucleases,enzymes that catalyzed the cleavage of DNA at specificsequences. The isolated DNA fragment was then heated todenature the double-stranded helix into two single-stranded

UNLEASHING THE POWER OFEXPONENTIAL GROWTH—THEPOLYMERASE CHAIN REACTION

In the early 1980s the fruits of the molecular biology revolution were beginning

to be realized. Geneticists were uncovering the genetic defects that lead to many

hereditary diseases, and the newly burgeoning biotechnology industry was eager

to provide physicians with simple diagnostic tests for such diseases. However, the

best method available for detecting abnormal genes, Southern hybridization,

required sizable DNA samples and several days to perform. In this environment,

one of the most powerful molecular biology techniques known was born: the

polymerase chain reaction, or PCR.


DNA molecules. At this point, the oligonucleotide wasadded to the DNA and allowed to anneal to the comple-mentary region, thereby creating a primer-template com-plex, one of the substrates for DNA polymerase. Theother substrates, the four deoxynucleotide triphosphates(dNTPs), were then added, so that DNA synthesis couldoccur. Although this method was useful for producingradioactively labeled pieces of DNA, it could not amplifya DNA sequence, only replicate it.

The ExperimentMullis designed a method that would actually amplify theamount of target DNA, a prerequisite for detecting a smallDNA sequence within a large complex sample of genomicDNA. For instance, the human genome conations nucleotides of coding sequence. Molecular diagnosis ofsickle cell anemia requires the detection of one alterednucleotide in one gene amongst the rest of the genome. Toaccomplish this, the region of the genome containing thealteration must be amplified.

Based on the sequence of the �-globulin gene, whichwas known, Mullis designed primers that would anneal at

3 � 109


sequences both upstream and downstream from the dis-ease causing mutation. One primer was complementary tothe coding strand, known as the ( ) strand, the secondwas complementary to the noncoding, or (�), strand.When the primers were added to a sample of denaturedgenomic DNA along with DNA polymerase and the fourdNTPs, DNA synthesis occurred across the region of themutation from both of the original strands, producing twonew double-stranded DNA molecules. Thus the DNAbetween the primer sites was doubled, not simply repli-cated as in the older method. Mullis realized that eachcycle of DNA-primer annealing and DNA synthesis wouldyield twice as much target DNA as the previous cycle (see Figure). A chain reaction would ensue and theamount of DNA in the sample would grow exponentially.

�

He called his technique the polymerase chain reaction(PCR) to reflect the mechanism by which amplificationwas occurring.

The first published test of the PCR made use ofupstream and downstream oligonucleotide primers thatflanked a 110-bp region of the �-globin gene; the targetregion included the mutation found in sickle cell anemia.These primers were mixed with samples of amniotic fluidthat had been previously typed for the presence or absenceof the mutation. After the samples were put through 20cycles of heat denaturation, cooling to allow annealing,and DNA synthesis or primer elongation, the amount of �-globin target DNA in the samples was found to beenriched more than one million times (220) compared withthe initial samples. The exponential expansion of the DNA was easily demonstrated by comparing the samesample after 15 and 20 cycles. It was clear that the addi-tional five cycles greatly increased the amount of DNAproduced in the reaction. Next, Mullis tested the ability ofthe PCR to detect small quantities of DNA. He found thatafter 20 cycles, the �-globulin gene could be detectedstarting with a genomic DNA sample as small as 20 ngwhich was 50 times smaller than the samples in the origi-nal tests. This finding implied that the PCR could be usedin a variety of situations where only a small amount ofDNA was available, contributing to the widespread use ofthe technique today.

DiscussionDevelopment of the PCR relied on two key insights.

First, that a DNA sequence could be amplified, not justreplicated, if synthesis were carried out from both the cod-ing and noncoding strands. Second, that a target DNAsequence would “grow” like dividing bacteria in a cultureif the amplification cycle was repeated several times insuccession. By employing this relatively simply methodol-ogy, Mullis developed one of the most powerful tech-niques in molecular biology.

The advantages of PCR were obvious from the firstreport. Almost instantly, it became a standard techniqueused in all fields of biology and medicine, as well as theforensic sciences. Today, the technique is known not onlyto biologists, but also to people in all walks of life. In1993, just eight years after his first report on the PCR,Kary Mullis was awarded the Nobel Prize for Chemistryfor developing this revolutionary technique.

5'3'

3'5'

3'3'

Add DNA polymerase [ ]dNTPs

Heat denatureCool to allow primer annealingRepeat reaction

Repeat reaction for 20 cycles

DNA encoding the β-globin gene

Upstream primer

Downstream primer

*

*

*

**

*

*

*

*

*

Nucleotide mutatedin sickle cell anemia

*

DenatureAdd primers in excess

Schematic of the polymerase chain reaction (PCR) to amplify the�-globin gene. In this case, one oligonucleotide primer is comple-mentary to the (�) strand and hybridizes downstream of themutation that leads to sickle cell anemia; the other primer is com-plementary to the (�) strand and hybridizes upstream of themutation. Repeated cycles of DNA denaturation, primer annealing,and DNA synthesis amplify the target sequences between theprimer-binding sites.


BackgroundAt the time of Hamilton Smith’s work, host restriction wasa well-characterized, yet highly intriguing phenomenon. Itwas well known that DNA from one species of bacteriacould not be used to transform a second species of bacte-ria. When researchers simply mixed DNA from one bacte-ria with a lysate from a second bacterial species, the DNAwas cleaved. The bacteria had evolved a system to recog-nize and cleave foreign DNA. In 1965, Werner Arberhypothesized that bacteria must produce an enzyme capa-ble of recognizing and cleaving foreign DNA at specificsequences. How did a bacterium determine which DNAwas foreign, and which was its own? It seemed unlikelythat a bacterium could exclude specific sequences in itsgenome, from the action of this nuclease. More likely, abacterium somehow modified its own DNA at thesesequences, so it could be spared from cleavage. The exis-tence of a second enzyme was thus hypothesized, one thatcould modify the DNA by methylation at the site wherecleavage occurred, thereby preventing cleavage by thesequence-specific nuclease.

With these hypotheses in hand, the hunt for theenzymes could begin. In 1968, Mathew Meselson reportedthe purification from E. coli of one of these enzymes nowcalled restriction enzymes or restriction endonucleases.Although the E. coli enzyme catalyzed the cleavage of

DEMONSTATING SEQUENCE-SPECIFICCLEAVAGE BY A RESTRICTION ENZYME

Bacteria exhibit a phenomena, known as host restriction, whereby they can

both recognize and cleave foreign DNA, preventing it from interfering with

the bacterial life cycle. By purifying and characterizing one of the enzymes

involved in host restriction, Hamilton Smith gave molecular biology one of its

most important tools, an enzyme that cleaves DNA at a specific sequence.


non-E.coli DNA, Meselson could not demonstrate thatthis cleavage was sequence specific. In fact, proving thatthese bacterial enzymes cleave DNA at a specific sequencewould be a tricky manner, as this research was conductedbefore the advent of the relatively simple DNA-sequencingtechniques now available. Following on Messelson’swork, Smith set out to purify a second restriction enzyme,this time from H. influenzae, and to demonstrate that itdoes indeed cleave DNA in a sequence-specific manner.

The ExperimentThe first step in the successful purification of a newenzyme is devising an assay that measures the knownactivity of the enzyme as it is being purified. The activityof a restriction enzyme is to catalyze the cleavage of for-eign DNA, so this was the logical activity to monitor. Todo so, Smith took advantage of the fact that genomicDNA from bacteria is quite viscous, however as nucleasesbegin to degrade the bacterial DNA, its the overall viscos-ity decreases. Therefore, Smith could monitor the purifi-cation of his restriction enzyme by measuring the decreasein viscosity of a foreign DNA after treatment with a sam-ple of the protein after each step in the purificationscheme. Smith mixed cell extracts of H. influenzae withintact DNA from either H. influenzae or the Salmonella


bacteriophage P22. Using a device called a viscometer, hemeasured how the DNA from P22 became less viscousover time, while the H. influenzae DNA displayed nochange in viscosity. This would be the assay he would usethroughout the purification scheme.

Smith used a variety of established methods to separatebacterial lysates into smaller pools of proteins. Eachmethod separated the lysate based on a different physicalproperty of the proteins (and other biomolecules) thatmake up the lysate. This allowed the lysate to be dividedinto subsamples known as fractions. After each step in thepurification, every fraction was separately assayed for theability to cleave P22 DNA. Fractions that contained theenzyme activity were subjected to yet another purificationmethod, and the process was continued until a pureenzyme was obtained. Smith called the purified restrictionenzyme endonuclease R.

Next Smith determined some of the basic characteris-tics of endonuclease R. He used endonuclease R to digestDNA from the bacteriophage T7, then estimated the num-ber of sites where the DNA was cleaved. He discoveredthat endonuclease R did not completely degrade T7 DNA,but rather cleaved it at approximately 40 sites. Since T7DNA contains approximately 40,000 bases, cleavageoccurred at only 0.1 percent of the possible sites. Thisobservation suggested to Smith that Arber’s hypothesiswas correct—the enzyme was cleaving the DNA at spe-cific sequences. In order to prove that this was the case,Smith had to determine the sequence at which the enzymecleaved the DNA, which he called the recognition site.

With the purified enzyme and evidence of sequence-specific DNA cleavage, Smith focused his attention ondetermining the sequence of the recognition site. At thistime, the 1960s, the only known method of DNA sequenc-ing was to sequentially remove nucleotides from the 5end of DNA and determine their identity by thin layerchromatography (TLC). Smith devised a scheme tosequence the recognition site by using known enzymes tocleave the ends of a DNA strand into small pieces thatcould be analyzed by TLC (see Figure).

Smith began by labeling the 5 end of endonuclease R-digested DNA with a radioactive marker, 32P. This wasaccomplished by first treating the DNA with alkaline phos-phatase, an enzyme that catalyzes the removal of 5 phos-phate groups from polynucleotides. Next, polynucleotidekinase, which catalyzes addition of phosphate to the 5 endof polynucleotides, was used to transfer 32P from labeledATP to the terminal nucleotide. Now, the terminalnucleotide could be easily distinguished from the rest of thenucleotides, by virtue of its specific radioactive label. TheDNA was then digested to single nucleotides with a nucle-ase called pancreatic DNase. The only 32P-labelednucleotides observed contained adenine (A) and guanine(G). Since no 32P-labeled nucleotide containing cytosine (C)

¿

¿

¿

¿

or thymine (T) was detected, Smith deduced that the firstbase in the recognition sequence must be a purine.

To determine the second base in the recognition site,Smith used a nuclease that could not cleave 5 terminal di-nucleotides. In other words, the entire DNA sample wasdigested into single nucleotides except the final two, whichremained in dinucleotide form. Since the DNA previouslyhad been cleaved with endonuclease R, the 5 terminal di-nulceotides are the first two bases in the recognition site.Smith first separated the dinucleotides from the singlenucleotides. When he analyzed the dinucleotides by TLC,he found only two species of dinucleotides that carried the32P label. The identity of the 32P-labeled dinucleotides wasdetermined by comparing their migration to that of dinu-cleotides of known sequence. One of the species displayedthe same migration as the dinucleotide GA; the othermigrated with the dinucleotide AA. Smith concluded thatthe second base in the recognition sequence was adenine.

Analysis of the rest of the recognition site would not beso easy, but Smith’s persistence paid off. He identified thethird base in the recognition site as cytosine using a similar,but slightly more complicated method. He further showedthis to be the end of the recognition sequence by showing

¿

¿

5'3'

5'3'

3'5'

3'5'

Endonuclease R

Alkaline phosphatase

Polynucleotide kinase[32P] ATP

Digestion with variousnucleases

PP

P**P

5'3'

3'5'

5'3'

3'5'

P* *P

*P

*P

Recognition site

Mononucleotides

Dinucleotides

Trinucleotidesn = 3

P*n = 2

P*n = 1

Schematic representation of the method used to determine thenucleotide sequence recognized by endonuclease R. T7 bacterio-phage DNA was digested with endonulcease R. After removal ofthe 5’ phosphate, and addition of a 32P label, the 5’ end-labeledDNA was digested with a variety of nucleases. 32P-labeledmononucleotides, dinucleotides, and trinucleotides were isolatedand analyzed to determine the recognition site sequence.[Adapted from T. J. Kelly and H. O. Smith, 1970, J. Mol.Biol.51:393.]


that the fourth nucleotide could contain any base. Now heknew digestion of double stranded DNA with endonucleaseR creates several smaller fragments with identical 5’ ends,which contain the sequence purine-adenine-cytosine. Sincethe DNA strands are complementary, the only possible waythis could occur is if the enzyme recognized a six-basesequence that appeared the same on either strand, known asa pallindromic sequence. Therefore, Smith concluded thatendonuclease R recognized and cleaved DNA specifically atthe sequence GTPyPuAC.

DiscussionAlthough the first restriction enzyme had been purifiedtwo years before Smith reported his work on endonucle-ase R, he was the first to demonstrate sequence-specific

cleavage. He then went on to purify and characterize themethylase that allows DNA from H. influenzae to escapecleavage. By using these sequence-specific restrictionenzymes, researchers could now cleave DNA at specificsites. The impact of restriction enzymes on biologicalresearch over cannot be overstated. Early on, theseenzymes were used for mapping plasmid and phage DNA.Now they are routinely used for probing the structure ofboth specific genes and of DNA from individuals. In addi-tion, they are primary reagents in the construction of geneexpression vectors, allowing DNA from different sourcesto be cleaved at specific sequences, then joined with simi-larly cleaved DNA. The results are seen everyday in labo-ratories employing recombinant DNA technologies. In1978, Hamiliton Smith was awarded the Nobel Prize forPhysiology and Medicine in recognition of his powerfuldiscovery.


BackgroundA powerful approach to the study of genes and the pro-teins they encode is the controlled expression in both cellsand whole organisms. Before the advent of recombinantDNA techniques, biologists accomplished this by injectingforeign mRNA into oocytes from frogs and studying thebiological activity of the protein encoded by the foreignmRNA. In the 1970s and 1980s, the molecular biologyrevolution allowed genes to be fused to specific promot-ers, which would allow them to be expressed in cell line.Whereas biologists became able to study the gene func-tion in cultured cells, they still wanted to study genes ina living organism. This requires the expression of a spe-cific foreign gene in embryonic cells, leading to introduc-tion of the foreign gene into the animal’s genome, and ex-amination of its function in the organism.

In the early 1970s, Brinster demonstrated that foreigngenes could be expressed in mice by injecting cancer cellsinto an early embryonic form of a developing mouseknown as a blastocyst. This approach, however, made itdifficult to express a specific gene in the desired cell types.This would require introducing the gene into the mousegenome. In 1980, biologists demonstrated that this waspossible by injecting a plasmid containing viral DNA intofertilized mouse oocytes, then detecting the viral sequencesin the newborn mice. This set the stage to determine

EXPRESSING FOREIGN GENES IN MICE

Bn the span of three years from 1980 – 1982, the notion of expressing foreign

proteins in mice went from an idea to a reality. During this time, several lab-

oratories worked furiously to introduce new genes and express exogenous pro-

teins, first in mouse embryonic stem cells and then in full-grown mice. Ralph

Brinster and Richard Palmiter were among the pioneers in this field when, in

1981, they first demonstrated the robust expression of a viral gene in a transgenic

mouse.


whether a functional protein could be expressed from aforeign gene incorporated into the mouse genome.

The ExperimentBrinster’s challenge was to design the experiment in sucha way that it could be easily and unequivocally demon-strated that the mouse was making the foreign protein. Toaccomplish this, Brinster chose to express an easily assayedenzyme rather than a protein of greater biological inter-est in his first transgenic mouse. He chose the enzymethymidine kinase from the herpes simplex virus (HSV), thechoice of which offered several advantages. First, the genecame from a human virus; thus its sequence sufficientlydiffered from the endogenous mouse gene allowing its in-tegration into the mouse genome to be readily demon-strated. Second, the activity of thymidine kinase can beeasily assayed by following the conversion of radioactivelylabeled thymidine to thymidine monophosphate. Finally,an inhibitor of the HSV thymidine kinase activity that doesnot inhibit the endogenous mouse enzyme was available,allowing the researchers to specifically monitor the activ-ity of the foreign protein.

Genes are expressed from DNA sequences upstream ofthe protein-coding region called promoters. Promoterscontrol where and when a gene is expressed. To express


a viral gene in a mouse requires that the biologist removethe gene from the control of the viral promoter and fuseit to a promoter that is active in mouse cells. Brinster col-laborated with Palmiter, who had been studying the pro-moter of the mouse metallothionein-1 (MT-1) gene.Palmiter fused the MT-1 promoter to the HSV thymidinekinase gene. They then could ask whether a viral proteincould be expressed in a mouse.

To generate the transgenic mouse, Brinster andPalmiter injected the plasmid containing HSV thymidinekinase fused to the MT-1 promoter into the pro-nuclei offertilized mouse eggs, which they then implanted back intofemale mice. The scientists mated progeny mice with nor-mal females, and analyzed the resulting progeny for thepresence of the HSV thymidine kinase DNA as well asthymidine kinase activity.

Using Southern blot analysis, they detected the pres-ence of the MT-1 promoter/thymidine kinase gene fusion,known as the transgene. They isolated genomic DNA, thencleaved it with a restriction endonuclease. They proceededto separate the DNA by agarose gel electrophoresis—which separates DNA fragments on the basis of size—and transferred it to a nitrocellulose membrane. The twoscientists then hybridized a radioactively labeled probe,specific for the transgene, to the membrane for analysis.This analysis revealed that the transgene had been suc-cessfully integrated into the genomes of four progeny mice.

Next, to determine whether the transgene expressed afunctional protein, Brinster and Palmiter analyzed ho-mogenates from the liver, a tissue where the mouse MT-1gene is highly expressed, for viral thymidine kinase activ-ity. Liver homogenates from one mouse contained ap-proximately 200 times more thymidine kinase activitythan the liver homogenates of its littermates. This mousewas one of the four that had the transgene integrated intoits genome. To demonstrate that this increase in activitywas a result of viral thymidine kinase expression theytreated liver homogenates with an inhibitor that specifi-cally blocks the HSV thymidine kinase activity. Thymidinekinase activity in liver homogenates from the transgenicmouse was markedly reduced by this inhibitor, whereas

the activity in homogenates from its non-transgenic litter-mates was unchanged (Table 8.1). Thus Brinster andPalmiter confirmed the presence of viral thymidine kinaseactivity, and demonstrated that a foreign protein could beexpressed in a mouse.

DiscussionProgress in embryology and molecular biology had left thefield ripe for researchers to experiment with advancing theexpression of foreign proteins in animals. The carefulchoice of the easily assayed HSV thymidine kinase geneput under the control of the metallothionein promoter al-lowed Brinster and Palmiter to demonstrate the feasibilityof this technique.

The ability to generate transgenic mice has been in-valuable to the study of gene function in vivo. Before thistechnology was available, researchers had to find natu-rally occurring mutations in order to analyze gene func-tion in mice. Now, a specific gene can be expressed inmice. Soon, genes were fused to promoters that allowedexpression in specific tissues. Scientists have generatedtransgenic mice to analyze the function of a great numberof genes, allowing them to determine the roles of the genesin a variety of diseases and biological processes.

TABLE 9-1 Expression of Viral Thymidine Kinasein Transgenic Mice

Mouse Transgene DNA Thymidine Kinase Activity

�Inhibitor �Inhibitor

23-1 � 14500 1470023-2 � 497,000 187,000

[Adapted from R. L. Brinster et al., 1981, Cell 27:223–231.]

8945d_027-028 6/20/03 1:01 PM Page 28 mac117 mac117:1268tm:8945d:

Background Research on the structure of Ig molecules provided someclues about the generation of antibody diversity. First, itwas shown that an Ig molecule is composed of fourpolypeptide chains: two identical heavy (H) chains andtwo identical light (L) chains. Some researchers proposedthat antibody diversity resulted from different combina-tions of heavy and light chains. Although somewhatreducing the number of genes needed, this hypothesis stillrequired that a large portion of the genome be devoted toIg genes. Protein chemists then sequenced several Ig lightand heavy chains. They found that the C-terminal regionsof different light chains were very similar and thus weretermed the constant (C) region, whereas the N-terminalregions were highly variable and thus were termed the

TWO GENES BECOME ONE—SOMATICREARRANGEMENT OFIMMUNOGLOBULIN GENES

For decades, immunologists wondered how the body could generate the multi-

tude of pathogen-fighting immunoglobulins, called antibodies, needed to ward

off the vast array of different bacteria and viruses encountered in a lifetime.

Clearly, these protective proteins, like all proteins, somehow were encoded in the

genome. But the enormous number of different antibodies potentially produced

by the immune system made it unlikely that individual immunoglobulin (Ig) genes

encoded all the possible antibodies an individual might need. In studies beginning

in the early 1970s, Susumu Tonegawa, a molecular biologist, laid the foundation

for solving the mystery of how antibody diversity is generated.


variable (V) region. The sequences of different heavychains exhibited a similar pattern. These findings suggestedthat the genome contains a small number of C genes anda much larger group of V genes.

In 1965, W. Dryer and J. Bennett proposed that twoseparate genes, one V gene and one C gene, encode eachheavy chain and each light chain. Although this proposalseemed logical, it violated the well-documented principlethat each gene encodes a single polypeptide. To avoid thisobjection, Dryer and Bennett suggested that a V and Cgene somehow were rearranged in the genome to form asingle gene, which then was transcribed and translatedinto a single polypeptide, either a heavy or light Ig chain.Indirect support for this model came from DNAhybridization studies showing that only a small number ofgenes encoded Ig constant regions. However, until more


powerful techniques for analyzing genes came on thescene, a definitive test of the novel two-gene model wasnot possible.

The ExperimentTonegawa realized that if immunoglobulin genes under-went rearrangement, then the V and C genes were mostlikely located at different points in the genome. The dis-covery of restriction endonucleases, enzymes that cleaveDNA at specific sites, had allowed some bacterial genes tobe mapped. However, because mammalian genomes aremuch more complex, he knew that similar mapping of thegenes encoding V and C regions was not technically feasi-ble. Instead, drawing on newly developed molecular biol-ogy techniques, Tonegawa devised another approach fordetermining whether the V and C regions were encoded bytwo separate genes. He reasoned that if rearrangement ofthe V and C genes occurs, it must happen during differen-tiation of Ig-secreting B cells from embryonic cells.Furthermore, if rearrangement occurs, there should bedetectable differences between unrearranged germ-lineDNA from embryonic cells and the DNA from Ig-secret-ing B cells. Thus, he set out to see if such differences existedusing a combination of restriction-enzyme digestion and RNA-DNA hybridization to detect the DNA frag-ments.

150

200

250

cpm

50

0

100

5 10 15

MIGRATION (cm)Top ofgel

Bottomof gel

Embryo DNA

Whole gene3' end of gene

Whole gene3' end of gene

150

200

250

cpm

50

0

100

5 10 15

MIGRATION (cm)Top ofgel

Bottomof gel

B Cell DNA

Experimental results showing that the genes encoding the variable (V) and constant (C) regions of k light chains are rearranged duringdevelopment of B cells. These curves depict the hybridization of labeled RNA probes, specific for the entire k gene (V � C) and for the 3’end that encodes the C region, to fraction of digested DNA separated by agarose gel electrophoresis. [Adapted from N. Hozumi and S.Tonegawa, 1976, Proc. Nat’l. Acad. Sci. USA 73:3629.]

He began by isolating genomic DNA from mouseembryos and from mouse B cells. To simplify the analysis,he used a line of B-cell tumor cells, all of which producethe same type of antibody. The genomic DNA was thendigested with the restriction enzyme BamHI, which recog-nizes a sequence that occurs relatively rarely in mam-malian genomes. Thus, the DNA was broken into manylarge fragments. He then separated these DNA fragmentsby agarose gel electrophoresis, which separates biomole-cules on the basis of charge and size. Since all DNA car-ries an overall negative charge, the fragments were sepa-rated based on their size. Next, he cut the gel into smallslices and isolated the DNA from each slice. Now,Tonegawa had many fractions of DNA pieces of varioussizes. He then could analyze these DNA fractions to deter-mine if the V and C genes resided on the same fragment inboth B cells and embryonic cells.

To perform this analysis, Tonegawa first isolated fromthe B-cell tumor cells the mRNA encoding the major typeof Ig light chain, called . Since a RNA is complementaryto one strand of the DNA from which it is transcribed, itcan hybridized with this strand forming a RNA-DNAhybrid. By radioactively labeling the entire mRNA,Tonegawa produced a probe for detecting which of theseparated DNA fragments contained the k chain. He thenisolated the 3 end of the mRNA and labeled it, yieldinga second probe that would detect only the DNA sequencesencoding the constant region of the k chain. With these

k¿

k

k


probes in hand—one specific for the combined V � Cgene and one specific for C alone—Tonegawa was readyto compare the DNA fragments obtained from B cells andembryonic cells.

He first denatured the DNA in each of the fractionsinto single strands and then added one or the other labeledprobe. He found that the C-specific probe hybridized todifferent fractions derived from embryonic and B-cellDNA (Figure 1). Even more telling, the full-length RNAprobe hybridized to two different fractions of the embry-onic DNA, suggesting that the V and C genes are not con-nected and that a cleavage site for BamHI lies betweenthem. Tonegawa concluded that during the formation of B cells, separate genes encoding the V and C regions arerearranged into a single DNA sequence encoding theentire light chain (Figure 2).

DiscussionThe generation of antibody diversity was a problem await-ing the molecular techniques to answer it. Tonegawa wenton to clone V-region genes and prove that the rearrange-ment must occur somatically. These findings impactedgenetics as well as immunology. Where once it wasbelieved that every cell in the body contained the samegenetic information, it became clear that some cells takethat information and alter it to suit other purposes. In

k

addition to somatic rearrangement, Ig genes undergo avariety of other alterations that allow the immune systemto create the diverse repertoire of antibodies necessary toreact to any invading organism. Our current understand-ing of these mechanisms rests on the foundation ofTonegawa’s fundamental discovery. For this work, hereceived the Nobel Prize for Physiology and Medicine in1987.

5' 3'VK CK

5' 3'VK CK

Embryonic DNA

B-Cell DNA

Schematic diagram of light-chain DNA in embryonic cells and Bcells that is consistent with Tonegawa’s results. In embryoniccells, cleavage with the BamHI restriction enzymes (red arrows)produces two different sized fragments, one containing the Vgene and one containing the C gene. In B cells, the DNA isrearranged so that the V and C genes are adjacent, with no inter-vening cleavage site. BamHI digestion thus yields one fragmentthat contains both the V and C genes.

k


BackgroundIn eukaryotes and many viruses, genes contain sequencesthat are initially transcribed, then subsequently removedfrom RNA, as they are not part of the actual codingsequence. These sequences are known as interveningsequences (IVS) or introns. IVS are removed from precur-sor RNA by a biological process known as splicing. Whileinvestigating the splicing of precursor ribosomal RNA(pre-rRNA) genes, transcribed from rRNA genes, Cechmade his critical discovery that RNA exhibited catalyticactivity.

Cech wanted to understand the molecular componentsof RNA splicing. Rather than examing complex eukary-otic genes, he chose a simple model system, rRNA genesfrom the ciliated protozoa Tetrahymena thermophilia. Byisolating Tetrahymena nuclei, Cech and his coworkersdeveloped a system in which pre-rRNA gene splicingcould be studied in vitro. The purified nuclei could per-form both transcription of rRNA genes and processing ofthe large pre-rRNA that initially is formed. Using this sys-tem, Cech found that during synthesis of 26s rRNA inTetrahymena, a 0.4-kb IVS is removed. The next step wasto perform pre-rRNA splicing with nuclear extracts, withan eye toward purifying the enzymes that catalyzed thesplicing reaction. Although Cech succeeded in this goal, he

CATALYSIS WITHOUT PROTEINS—THEDISCOVERY OF SELF-SPLICING RNA

For biological systems to function, countless reactions must be catalyzed. These

duties are carried out by enzymes, biological macromolecules that readily

enhance reaction rates yet remain unconsumed by the reaction. For many years

only proteins were believed to possess sufficient diversity of functional groups to

catalyze the myriad reactions necessary to sustain life. Then in 1981, Thomas

Cech reported that, in at least one case, RNA could do the job.


could have never guessed how the catalysis was takingplace.

The ExperimentCech’s plan was to use the in vitro splicing system to purifythe RNA-splicing enzymes, a common experimentalapproach for dissecting complex molecular processes.First, the reaction is characterized in a cell-free system, inthis case purified nuclei. Then a means to purify the reac-tion substrate is developed. In the case of the TetrahymenarRNA splicing this was relatively easy, because the full-length rRNA (pre-rRNA) transcripts were abundant inTetrahymena nuclei and readily purified. Finally, cellularextracts are added back to reconstitute the activity beingstudied. Since the RNA splicing activity was known totake place in the nucleus, Cech used nuclear extracts. Infact, he could readily see splicing when nuclear extractswere added to rRNA transcripts in a splicing cocktailcomposed of Mg2� and guanosine triphosphate (GTP).Unexpectedly, splicing also occurred when rRNA tran-scripts were incubated in the splicing cocktail in theabsence of a nuclear extract. This activity was repro-ducible, leaving open two possibilities: Either the purifiedpre-rRNA remained associated with an enzyme (i.e., a


protein contaminant) or the pre-rRNA was catalyzing itsown splicing.

The first step in determining which possibility was cor-rect was to see if the rRNA transcripts were truly devoid ofprotein. Because proteins are notoriously fragile biomole-cules, whose activity is easily destroyed by heat, chemicals,and proteolytic enzymes, Cech subjected the rRNA tran-scripts to numerous treatments known to degrade proteins.First, boiling to promote heat denaturation. Then, extrac-tion with organic solvents to promote chemical denatura-tion. Finally, incubation with a variety of proteases to pro-mote enzymatic degradation. Still, the pre-rRNA retainedits splicing activity. These results strongly suggested thatTetrahymena pre-rRNA is indeed self-splicing. But a moredefinitive experiment was needed to convince otherresearchers that the transcripts were uncontaminated byprotein and possessed inherent catalytic activity.

Fortunately, the Tetrahymena pre-rRNA could be pro-duced in vitro using purified RNA polymerase from

E. coli. Transcription of the Tetrahymena rRNA gene witha polymerase from a different organism would eliminatethe risk that the RNA remained associated with aTetrahymena enzyme. In this system, the only enzyme everassociated with RNA would be E.Coli RNA polymerase,which was readily removed by extraction with organic sol-vents. Using this system, Cech carefully synthesized theTetrahymena pre-rRNA, removed the polymerase, andpurified the transcripts. When he incubated this in vitrosynthesized pre-rRNA in the splicing cocktail, analysis ofthe products showed that once again, the IVS wasremoved from the precursor (see Figure). This experimentproved that the Tetrahymena pre-RNA was self-splicing,catalyzing the removal of the IVS without the aid of anyprotein.

DiscussionCech called his self-splicing RNA a “ribozyme,” implyingthat it was an RNA enzyme. Although the demonstrationof self-splicing RNA was readily accepted by the scientificcommunity, many were skeptical about the notion thatRNA was a true catalyst. In subsequent studies, however,Cech was able to engineered the Tetrahymena rRNA IVSsuch that it could be used as an enzyme, splicing one RNAmolecule, then turning over to splice others. This con-vinced even the skeptics that RNA can have true catalyticactivity. Soon, other self-splicing RNAs and other cata-lytic RNAs were identified. RNA catalysis has become a field of study unto itself, with research on the use of cat-alytic RNA in both laboratory and medical settings.Furthermore, the ability of RNA to catalyze biologicalreactions has evolutionary implications. It is now conceiv-able that primordial organisms contained only RNA andlater evolved the more complex system of proteins. For hispioneering work on RNA catalysis, Cech was awarded theNobel Prize for Chemistry in 1989.

IVS

Pre-rRNA alone Pre-rRNA + Mg2+ and GTP

Demonstration that Tetrahymena thermophilia pre-rRNA can self-splice. Radioactively labeled pre-rRNA was synthesized in vitrousing E. coli RNA polymerase and then incubated in neutral bufferor in the presence of Mg2� and GTP, necessary cofactors for thesplicing reaction. Depicted here is an autoradiograph of the elec-trophoresed samples revealing the spliced-out IVS in the samplecontaining splicing cofactors. [Adapted from K. Kruger et al., 1982,Cell 31:147.]


BackgroundThe discovery of GTP’s role in regulating signal transduc-tion began with studies on how glucagon and other hor-mones send a signal across the plasma membrane thateventually evokes a cellular response. At the outset ofRodbell’s studies, it was known binding of glucagon tospecific receptor proteins embedded in the membranestimulates production of cAMP. The formation of cAMPfrom ATP is catalyzed by a membrane bound enzymecalled adenyl cyclase. It had been proposed that the actionof glucagon, and other cAMP stimulating hormones,relied on additional molecular components that couplereceptor activation to the production of cAMP. However,in studies with isolated fat cell membranes known as“ghosts,” Rodbell and his coworkers were unable to pro-vide any further insight into how glucagon binding leadsto an increase in production of cAMP. Rodbell then begana series of studies with a newly developed cell-free system,purified rat liver membranes, which retained both mem-brane-bound and membrane-associated proteins. Theseexperiments eventually led to the finding that GTP isrequired for the glucagon-induced stimulation of adenylcyclase.

THE INFANCY OF SIGNALTRANSDUCTION—GTP STIMULATION OF CAMP SYNTHESIS

In the late 1960s the study of hormone action blossomed following the discovery

that cyclic adenosine monophosphate (cAMP) functioned as a second messanger,

coupling the hormone-mediated activation of a receptor to a cellular response. In

setting up an experimental system to investigate the hormone induced synthesis of

cAMP, Martin Rodbell discovered an important new player in intracellular sig-

nalling — guanosine triphosphate (GTP)


The ExperimentOne of Rodbell’s first goals was to characterize the bind-ing of glucagon to the glucagon receptor in the cell-free ratliver membrane system. First, purified rat liver membraneswere incubated with glucagon labeled with the radioactiveisotope of iodine (125I). Membranes were then separatedfrom the unbound [125I]glucagon by centrifugation. Onceit was established that labeled glucagon would indeedbind to the purified rat liver cell membranes, the studywent on to determine if this binding led directly to activa-tion of adenyl cyclase and production of cAMP in thepurified rat liver cell membranes.

The production of cAMP in the cell-free systemrequired the addition of ATP, the substrate for adenylcyclase, Mg2�, and an ATP-regenerating system consist-ing of creatine kinase and phosphocreatine. Surprisingly,when he glucagon binding experiment was repeated in thepresence of these additional factors, Rodbell observed a50 percent decrease in glucagon binding. Full bindingcould be restored only when ATP was omitted from thereaction. This observation inspired an investigation of theeffect of nucleoside triphosphates on the binding ofglucagon to its receptor. It was shown that relatively high


(i.e., millimolar) concentrations of not only ATP but alsouridine triphosphate (UTP) and cytidine triphosphate(CTP) reduced the binding of labeled glucagon. In con-trast, the reduction of glucagon binding in the presence ofGTP occurred at far lower (micromolar) concentrations.Moreover, low concentrations of GTP were found to stim-ulate the dissociation of bound glucagon from the recep-tor. Taken together, these studies suggested that GTP altersthe glucagon receptor in a manner that lowers its affinityfor glucagon. This decreased affinity both affects the abil-ity of glucagon to bind to the receptor, and encourages thedissociation of bound glucagon.

The observation that GTP was involved in the actionof glucagon led to a second key question: Can GTP alsoexert an affect on adenyl cyclase? Addressing this questionexperimentally required the addition of both ATP, as asubstrate for adenyl cyclase, and GTP, as the factor beingexamined, to the purified rat liver membranes. However,the previous study had shown that the concentration ofATP required as a substrate for adenyl cyclase could affectglucagon binding. Might it also stimulate adenyl cyclase?The concentration of ATP used in the experiment couldnot be reduced, because ATP was readily hydrolyzed byATPases present in the rat liver membrane. To get around

this dilemma, Rodbell replaced ATP with an AMP analog,5 -adenyl-imidodiphosphate (AMP-PNP), that can be con-verted to cAMP by adenyl cyclase, yet is resistant tohydrolysis by membrane ATPases. The critical experimentnow could be performed. Purified rat liver membraneswere treated with glucagon both in the presence andabsence of GTP, and the production of cAMP from AMP-PNP was measured. The addition of GTP clearly stimu-lated the production of cAMP when compared toglucagon alone (see Figure) indicating that GTP promotesnot only the binding of glucagon to its receptor but alsothe activation of adenyl cyclase.

DiscussionTwo key factors led Rodbell and his colleagues to detectthe role of GTP in signal transduction, whereas previousstudies had failed to do so. First by switching from fat cell ghosts to the rat liver membrane system, the Rodbellresearchers avoided contamination of their cell-free sys-tem with GTP, a problem associated with the procedurefor isolating ghosts. Such contamination would mask theeffects of GTP on glucagon binding and actviation ofadenyl cyclase. Second, when ATP was first shown toinfluence glucagon binding, Rodbell did not simply acceptthe plausible explanation that ATP, the substrate foradenyl cyclase, also affects binding of glucagon. Instead,he chose to test the effects on binding of the other com-mon nucleoside triphosphates. Rodbell later noted that he knew commercial preparations of ATP often are contami-nated with low concentrations of other nucleoside triphos-phates. The possibility of contamination suggested to himthat small concentrations of GTP might exert large effects on glucagon binding and the stimulation of adenylcyclase.

This critical series of experiments stimulated a largenumber of studies on the role of GTP in hormone action,eventually leading to the discovery of G proteins, theGTP-binding proteins that couple certain receptors to theadenyl cyclase. Subsequently, an enormous family ofreceptors that require G proteins to transduce their signalswere identified in eukaryotes from yeast to man. These G protein–coupled receptors are involved in action of manyhormones as well as in a number of other biological activ-ities including neurotransmission and the immuneresponse. It is now known that binding of ligands to theircognate G protein–coupled receptors stimulates the asso-ciated G proteins to bind GTP. This binding causes trans-duction of a signal that stimulates adenyl cyclase to pro-duce cAMP and also desensitization of the receptor, whichthen releases its ligand. Both of these affects wereobserved in Rodbell’s experiments on glucagon action. Forthese seminal observations, Rodbell was awarded theNobel Prize for Physiology and Medicine in 1994.

¿

1000

800

600

400

200

00

Glucagon + GTP

Glucagon

Basal

pm

ols

cA

MP

Minutes

5 10 15 20

Effect of GTP on glucagon-stimulated cAMP production fromAMP-PNP by purified rat liver membranes. In the absence of GTP,glucagon stimulates cAMP formation about twofold over the basallevel in the absence of added hormone. When GTP also is added,cAMP production increases another fivefold. [Adapted from M.Rodbell et al., 1971, J. Biol. Chem. 246:1877.]


BackgroundThe discovery of nitric oxide as a signaling molecule beganwith studies on the mechanism by which blood vesselsrelax and constrict, processes known as vasodilation andvasoconstriction. In addition to their desire to understandthe basic biology of these processes, scientists recognizedits medical importance, as drugs that promote vasodilationcould aid in the treatment of cardiovascular diseases.Nitroglycerin, long used to treat angina pectoris, wasknown to promote vasodilation. When applied to isolatedblood vessels, nitroglycerin and other nitrogen-containingcompounds had been found to activate a signalingpathway that began by stimulating the production of cyclicguanosine monophosphate (cGMP), and eventuallyresulted in dilation. There was much interest in discoveringthe natural signal for this process.

In vivo, vasodilation was known to occur after stimu-lation of vessels by the neurotransmitter acetylcholine.However, uncovering the mechanism of this response washindered by a puzzling finding by Robert Furchgott. In hisresearch on the constriction and relaxation of blood ves-sels, Furchgott was using isolated rabbit aorta as a modelsystem. He found that adding the neurotransmitter acetyl-choline to section of isolated rabbit aorta in vitro caused

SENDING A SIGNAL THROUGH A GAS

For decades scientists have tried to understand how cells work together in tis-

sues, as well as in whole organisms. By the 1980s, the identity of many signal-

ing molecules, the cellular responses they evoked, and many aspects of intracellu-

lar signaling pathways were understood. All the known signaling molecules — the

familiar hormones and neurotransmitters — were nongaseous substances, primari-

ly peptides and amino acid derivatives. However, studies on the dilation of blood

vessels showed that the gas nitric oxide (NO) could indeed function as a signaling

molecule.


the blood vessel to constrict, just the opposite of the nor-mal in vivo response. However, when he tried to repeatand expand these studies with another aorta preparation,a different response occurred. Now, adding acetylcholineto the aorta caused it to dilate, or relax. Trying to un-cover why the effect of acetylcholine was inconsistent,Furchgott discovered significant differences in the aortapreparations used in the two experiments.

In the body, blood vessels are made up of two types ofcells: smooth muscle cells that form the vessel wall, andendothelial cells, which line the inside wall facing the vessellumen. Furchgott found that when an isolated aorta prepa-ration contained endothelial cells as well as smooth musclecells, the vessel responded to acetylcholine by relaxing. Butwhen the endothelial cells were removed, vasoconstrictionwas once again seen with acetylcholine treatment. Toexplain these results, Furchgott proposed that acetylcholinecauses the endothelial cells to release a signaling moleculethat in turn causes smooth muscles to relax. Dubbing thisproposed molecule endothelium-derived relaxation factor,or EDRF, he set out to determine its nature and identity.Subsequent work by Furchgott and two other scientistswould reveal that nitric oxide is behind the drug-induceddilation of blood vessels but also the natural physiologicalprocess of vasodilation stimulated by acetlycholine.


The ExperimentsIn his search to identify EDRF, Furchgott initial tested theability of numerous classical signaling molecules to inducedilation of isolated aorta sections stripped of endothelialcells, his in vitro assay for EDRF activity. None of the var-ious hormones, prostaglandins, and cyclic nucleotides hetested exhibited EDRF activity. In 1986, Furchgott real-ized that the only molecule known to elicit vasodilation ofisolated blood vessels was nitroglycerin. It had been pos-tulated that the pharmacological action of nitroglycerin isdue to release of the gas nitric oxide (NO). Could the elu-sive EDRF actually be nitric oxide? To test this idea,Furchgott treated isolated blood vessels, stripped ofendothelial cells, with nitric oxide produced from acidifiedNaNO2. He found that the response of these stripped ves-sels to nitric oxide was similar to the dilation of isolatedvessels with their endothelium intact caused by the pro-posed EDRF release following acetylcholine treatment.This observation suggested he was on the right track. Hethen reasoned that if EDRF is nitric oxide, the same com-pounds should inhibit NO activity and EDRF activity.Subsequently, he showed that hemogloblin and other com-pounds that bind nitric oxide do indeed inhibit both NO-mediated dilation of stripped vessels and EDRF-mediateddilation of intact vessels. These findings led Furchgott tohypothesize that EDRF was nitric oxide. This hypothesiswas echoed by a second scientist, Louis Ignarro, whothrough similar reasoning and experimentation was led tothe same model.

Meanwhile, a third scientist, Salvador Moncada, inde-pendently conducted a critical set of experiments clearlydemonstrating that EDRF and nitric oxide elicit the identi-cal biological response and are inhibited by the same com-pounds. Moncada went on to show that the short half-lifeof both nitric oxide and EDRF is extended by adding theenzyme superoxide dismutase to the in vitro system. This

Some of the evidence supportimg the identity of EDRF and nitric oxide*

EDRF NO

Biological ResponseEffect on blood vessels in vitro Relaxation RelaxationStimulates cGMP production Yes Yes

Response to other agentsHemoglobin Inhibits relaxation Inhibits relaxationSuperoxide dismutase Extends half-life Extends half-life

*EDRF endothelial-derived relaxation factor, which is released from endothelial cells in response to acetylcholine.SOURCE: M. T. Kahn and R. Furchgott, 1987, in M. J. Rand and C. Raper, eds., Pharmacology, Elsevier Science Publisher, pp. 341–344; R. M. J.Palmer et al., 1987, Nature 327: 524; and L. J. Ignarro et al., Proc. Nat’l. Acad. Sci. USA 84: 9265.

�

enzyme catalyzes the conversion of oxygen free radicals,which would normally react with nitric oxide yieldingNO3

– and oxygen. Based on their identical biologicalresponses and susceptibilities to the same inactivatingagents, Moncada concluded that EDRF is nitric oxide.

The final proof that EDRF is indeed nitric oxide camein a paper published by Ignarro late in 1987. He had ear-lier reported biological and inhibitor data similar to thoseof Furchgott and Moncada (see Figure). However, he wenta step further, realizing that the only way to prove EDRFand nitric oxide were one and the same molecule would bethrough chemical identification. To do this, Ignarro treatedisolated blood vessels with acetylcholine, then collectedand chemically analyzed the surrounding medium. Hefound nitric oxide in the medium from vessels thatretained their endothelial cells, whereas no nitric oxidewas detectable in the medium surrounding stripped ves-sels. This evidence served as undeniable proof thatendothelial cells signaled vasodilation through the releaseof nitric oxide.

DiscussionWhile initially a startling and improbable hypothesis, therole of nitric oxide as a signaling molecule rapidly becamean exciting field of research. Soon after these criticalexperiments, Moncada went on to identify the enzymethat produces nitric oxide. In just a few short years, thisunusual signal was implicated in many other biologicalprocesses including neurotransmitter release and immunity.These exciting advances were predicated on the will-ingness of Furchgott and Ignarro’s to stretch the conceptof signaling molecules to include a gas that is unstable insolution. For this foresight, and the experiments resultingin the identification of EDRF as nitric oxide, they sharedthe Nobel Prize for Physiology and Medicine in 1998.


BackgroundIn addition to synthesizing proteins to carry out cellularfunctions, many cells must also produce and secrete addi-tional proteins that perform their duties outside of the cell.The mechanism of protein secretion fascinated many cellbiologists, including Palade. In early research on secretion,cells were disrupted and the various organelles separatedby centrifugation. These cell fractionation studies hadshown that secreted proteins are present in membrane-bound vesicles associated with the endoplasmic reticulm(ER), where they are synthesized, and with the plasmamembrane, where they are eventually released from thecell. Unfortunately, results from these studies were hard tointerpret due to difficulties in obtaining clean separationsbetween different organelles. To further clarify the path-way, Palade turned to a newly developed technique, high-resolution autoradiography, that allowed him to followradioactively labeled proteins as they moved within thecell. His work led to the seminal finding that secreted pro-teins travel within vesicles from the ER to the Golgi com-plex, and then to the plasma membrane.

FOLLOWING A PROTEIN OUT OF THE CELL

The advent of electron microscopy allowed researchers to see the cell and its

structures at an unprecedented level of detail. George Palade utilized this tool

not only to look at the fine details of the cell, but also to analyze the process of

secretion. By combining electron microscopy with pulse-chase experiments, Palade

uncovered the path proteins follow to leave the cell.


The ExperimentPalade wanted to identify which cell structures andorganelles participate in protein secretion. To study such acomplex process, he carefully chose an appropriate modelsystem for his studies, the pancreatic exocrine cell, whichis responsible for producing and secreting large amountsof digestive enzymes.

Palade first examined the protein secretion pathway invivo by injecting guinea pigs with [3H]-leucine, which wasincorporated into newly made proteins, thereby radioac-tively labeling them. At time points from 4 minutes to 15hours, the animals were sacrificed, and the pancreatic tis-sue was fixed. By subjecting the specimens to autoradiog-raphy and viewing them in an electron microscope, Paladecould trace where the labeled proteins were in cells at var-ious times. As expected, the radioactivity localized in vesi-cles at the ER at time points immediately following the[3H]leucine injection, and at the plasma membrane at thelater time points. The surprise came in the middle timepoints. Rather than traveling straight from the ER to theplasma membrane, the radioactively labeled proteins


appeared to stop off at the Golgi complex in the middle oftheir journey. In addition, there never was a time pointwhere the radioactively labeled proteins were not confinedto vesicles.

The observation that the Golgi complex was involvedin protein secretion was both surprising and intriguing. Tothoroughly address the role of this organelle in proteinsecretion, Palade turned to in vitro pulse-chase experi-ments, which permitted more precise monitoring of thefate of labeled proteins. In this labeling technique, cells are exposed to radiolabeled precursor, in this case

[3H]leucine, for a short period of time known as the“pulse.” The radioactive precursor is then replaced with itsnonlabeled form for a subsequent “chase” period. Proteinssynthesized during the pulse period will be labeled anddetected by autoradiography, while those synthesized duringthe chase period, being nonlabeled, will not be detected.Palade began by cutting guinea pig pancreas into thick slices,which were then incubated for 3 minutes in media contain-ing [3H]- leucine. At the end of the pulse, he added excessunlabeled leucine. The tissue slices were then either fixedfor autoradiography or used for cell fractionation. To

(c)

(a) (b)

(d)

The synthesis and movement of guinea-pig pancreatic secretory proteins as revealed by electron microscope autoradiography. (a) At theend of a 3-minute labeling period with [3H]leucine, the tissue is fixed, sectioned for electron microscopy, and subjected to auto-radiography. Most of the labeled proteins (the autoradiographic grains) are over the rough ER. (b) Following a 7-minute chase period withunlabeled leucine, most of the labeled proteins have moved to the Golgi vesicles. (c) After a 37-minute chase, most of the proteins areover immature secretory vesicles. (d) After a 117-minute chase, the majority of the proteins are over mature zymogen granules.[Courtesy of J. Jamieson and G. Palade.]


assure that his results were an accurate reflection of pro-tein secretion in vivo, Palade meticulously characterizedthe system. Once convinced that his in vitro system accu-rately mimicked protein secretion in vivo, he proceeded tothe critical experiment. He pulse-labeled tissue slices with[3H]leucine for 3 minutes, then chased the label for 7, 17,37, 57, and 117 minutes with unlabeled leucine. Radio-activity, again confined in vesicles, began at the ER, thentraveled in vesicles to the Golgi complex, and remained inthe vesicles as they passed through the Golgi and onto theplasma membrane (see Figure). As the vesicles traveledfarther along the pathway they became more denselypacked with radioactive protein. From his remarkableseries of autoradiograms at different chase times, Paladeconcluded that secreted proteins travel in vesicles from theER to the Golgi and onto the plasma membrane, and thatthroughout this process they remain in vesicles and do notmix with the rest of the cell.

DiscussionPalade’s experiments gave biologists the first clear look atproteins traveling through the secretory pathway. His

studies on the pancreatic exocrine cells yielded two semi-nal observations. First, that secreted proteins pass throughthe Golgi complex on their way out of the cell. This wasthe first function assigned to the Golgi complex. Second,secreted proteins never mix with other cellular proteins;they are segregated into vesicles throughout the pathway.These findings were predicated on two important aspectsof the experimental design. Palade’s careful use of electronmicroscopy and autoradiography allowed him to look atthe fine details of the pathway. Of equal importance wasthe choice of a cell type devoted to secretion, pancreaticexocrine cell, as a model system. In a different cell type,significant amounts of nonsecreted proteins might havealso been produced during the pulse, possibly leading toambiguous results.

Palade’s work set the stage for more detailed studies..Once the secretory pathway was clearly described, entirefields of research were opened up to investigation, in thesynthesis and movement of both secreted and membraneproteins. For this ground-breaking work, Palade wasawarded the Nobel Prize for Physiology and Medicine in1974.


BackgroundThe ability of muscles to perform work has long been afascinating process. Voluntary muscle contraction is per-formed by striated muscles, which are named for their ap-pearance when viewed under the microscope. By the1950s, biologists studying myofibrils, the cells that makeup muscles, had named many of the structures they hadobserved under the microscope. One contracting unit,called a sarcomere, is made up of two main regions calledthe A band, and the I band. The A band contains twodarkly colored thick striations and one thin striation. TheI band is made up primarily of light-colored striations,which are divided by a darkly colored line known as theZ disk. Although these structures had been characterized,their role in muscle contraction remained unclear. At thesame time, biochemists also tried to tackle this problemby looking for proteins that are more abundant in my-ofibrils than in other non-muscle cells. They found mus-cles to contain large amounts of the structural proteinsactin and myosin in a complex with each other. Actin andmyosin form polymers that can shorten when treated withadenosine triphosphate (ATP).

With these observations in mind, Hanson and Huxleybegan their study of cross striations in muscle. In a fewshort years, they united the biochemical data with the mi-croscopy observations and developed a model for musclecontraction that holds true today.

LOOKING AT MUSCLE CONTRACTION

The contraction and relaxation of striated muscles allow us to perform all of

our daily tasks. How does this happen? Scientist have long looked to see how

fused muscles cells, called myofibrils, differ from other cells that cannot perform

powerful movement. In 1954, Jean Hanson and Hugh Huxley published their

microscopy studies on muscle contraction, which demonstrated the mechanism by

which it occurs.


The ExperimentHanson and Huxley primarily used phase-contrast mi-croscopy in their studies of striated muscles that they iso-lated from rabbits. The technique allowed them to obtainclear pictures of the sarcomere, and to take careful mea-surements of the A and the I bands. By treating the mus-cles with a variety of chemicals, then studying them underthe phase-contrast microscope, they were able to success-fully combine biochemistry with microscopy to describemuscle structure as well as the mechanism of contraction.

In their first set of studies, Hanson and Huxley em-ployed chemicals that are known to specifically extract ei-ther myosin or actin from myofibrils. First, they treatedmyofibrils with a chemical that specifically removesmyosin from muscle. They used phase-contrast mi-croscopy to compare untreated myofibrils to myosin-extracted myofibrils. In the untreated muscle, they observedthe previously identified sarcometic structure, includingthe darkly colored A band. When they looked at themyosin-extracted cells, however, the darkly colored Aband was not observed. Next, they extracted actin fromthe myosin-extracted muscle cells. When they extractedboth myosin and actin from the myofibril, they could seeno identifiable structure to the cell under phase-contrastmicroscopy. From these experiments, they concluded thatmyosin was located primarily in the A band, whereas actinis found throughout the myofibril.


With a better understanding of the biochemical natureof muscle structures, Huxley and Hanson went on to studythe mechanism of muscle contraction. They isolated indi-vidual myofibrils from muscle tissue and treated them withATP, causing them to contract at a slow rate. Using thistechnique, they could take pictures of various stages ofmuscle contraction by using phase-contrast microscopy.They could also mechanically induce stretching by ma-nipulating the coverslip, which allowed them to also ob-serve the relaxation process. With these techniques inhand, they examined how the structure of the myofibrilchanges during contraction and stretch.

First, Huxley and Hanson treated myofibrils with ATP,then photographed the images they observed under phase-contrast microscopy. These pictures allowed them to mea-sure the lengths of both the A band and the I band at var-ious stages of contraction. When they looked at myofibrilsfreely contracting, they noticed a consistent shortening ofthe lightly colored I band, whereas the length of the Aband remained constant (see Figure 18.1). Within the A

band, they observed the formation of an increasingly densearea throughout the contraction.

Next, the two scientists examined how the myofibrilstructure changes during a simulated muscle stretch. Theystretched isolated myofibrils mounted on glass slides bymanipulating the coverslip. They again photographedphase-contrast microscopy images and measured thelengths of the A and the I bands. During stretch the lengthof the I band increased, rather than shortened, as it hadin contraction. Once again, the length of the A band re-mained unchanged. The dense zone that formed in the Aband during contraction, became less dense during stretch.

From their observations, Hanson and Huxley devel-oped a model for muscle contraction and stretch (see Fig-ure 18.1). In their model, the actin filaments in the I bandare drawn up into the A during contraction, and thus theI band becomes shorter. This allows for increased inter-action between the myosin located in the A band and theactin filaments. As the muscle stretches, the actin filamentswithdraw from the A band. From these data, they pro-posed that muscle contraction is driven by actin movingin and out of a mass of stationary myosin molecules.

DiscussionBy combining microscopic observations with known bio-chemical treatments of muscle fibers, Hanson and Huxleywere able to describe the biochemical nature of musclestructures and outline a mechanism for muscle contrac-tion. A large body of research continues to focus on un-derstanding the process of muscle contraction. Scientistsnow know that muscles contract by ATP hydrolysis driv-ing a conformational change in myosin that allows it topush actin along. Researchers are continuing to uncoverthe molecular details of this process, while the mechanismcontraction proposed by Hanson and Huxley remains inplace.

Stretched120%

Relaxed100%

Contracted90%

Contracted80%

Contracted60%

I bandsZ disk

A band

SAI

2.81.51.3

µ

µµ

SAI

2.31.50.8

µ

µµ

SAI

2.01.50.5

µ

µµ

SAI

1.81.50.3

µ

µµ

S 1.5µ

▲ FIGURE 19.1 Schematic diagram of muscle contraction

and stretch observed by Hanson and Huxley. The lengths ofthe sarcomere (S), the A band (A), and the I band (I) were mea-sured from 60 percent contraction (bottom) to 120 percentstretch (top). The lengths of the sacromere, the I band, and Aband are noted on the left. Notice that from 120 percent stretchto 70 percent contraction the A band does not change in thelength, whereas the length of the I band can stretch to 1.3 mi-crons, then contract to 0.3 microns. At 60 percent contraction,the I band disappears, and the A band shortens to the overalllength of the sarcomere. [Adapted from J. Hanson and H. E.Huxley, 1955, Symp. Soc. Exp. Biol. Fibrous Proteins and their Biological Significance 9:249.]


BackgroundNeurons are highly specialized cells that have several inter-esting features. A neuron has four main components: the cellbody, the dendrites, the axon, and the axon terminus. Thevast majority of proteins and membranes are synthesized inthe cell body. The axons extend from the cell body to theaxon terminus where neurotransmission is carried out. Pro-teins, membranes, and organelles are needed at the axon ter-minus for neurotransmission to occur. Therefore, there mustbe a system to transport biomolecules along the axon. In thelate 1960s, researchers took the first steps toward under-standing this system of transport by trying to characterizethe rate of transport. They found that radioactively labeledamino acids injected into ganglia could be taken up by thecell body of neurons and incorporated into proteins. Thisallowed the researchers to follow newly synthesized proteinsas they were transported to the axon terminus. Using thistechnique, Lasek and Ochs discovered that not all proteinstraveled along the axon at the same rate.

The ExperimentLasek and Ochs set out independently to study axonaltransport. To truly assess the rate of transport, each chose

RACING DOWN THE AXON

The field of neurobiology is filled with fascinating puzzles, but neurons also

pose interesting questions in the study of cell biology. It is well known that

neurons are extremely long cells stretching from the cell bodies in the central ner-

vous system into all areas of the body. But how does the neuron transport neuro-

transmitters and other biologically important molecules to the ends of the axon?

A piece to this puzzle was uncovered in the late 1960s when Raymond Lasek and

Sidney Ochs independently described fast axonal transport.


to study the sciatic nerve, which provides a long axon inwhich to study transport. Each of their experiments in-volved injecting radioactively labeled leucine ([3H]leucine)into the L7 dorsal root ganglia, the location of the cellbodies in the spinal cord. They analyzed transport alongthe axon by removing the nerve at various time points af-ter injection, sectioning the axon into small pieces, andthen measuring the amount of radioactivity by scintilla-tion counting. By following this protocol, Lasek and Ochswere each able to determine the rate of transport in theaxon.

Lasek devised a set of precautions to assure that themovement of radioactivity he observed was due to actualtransport and not to passive diffusion. The choice of a cellwith a long axon was one such precaution. He also per-formed a number of important controls. He used a com-bination of microscopy and autoradiography to demon-strate that [3H]leucine did not diffuse more than 2 mmfrom the injection site, but rather it was specifically takenup by the neuronal cell bodies and other cells in the area.He then tested whether the axon itself could take up theradioactively labeled amino acids by injecting [3H]leucinein the ventral root ganglia, an area devoid of cell bodies.If the axon took up radioactive amino acids, then Lasekwould find radioactivity as far away from the ventral rootinjection site as he had found in the dorsal root injectionsite. One day after injections, he found little radioactivity


more than 15 mm away in the ventral root, whereas hefound 40 times more in the dorsal root. These controls as-sured him that he was looking at the transport of ra-dioactively labeled leucine in the axon.

To characterize transport of radioactively labeledamino acids and/or proteins, Lasek measured the distrib-ution of radioactivity along the axon at time points rang-ing from 14 hours to 60 days after the initial injection of[3H]leucine. By six days, a large amount of the radioac-tivity had been transported out of the ganglia and downinto the axons. As expected, by 60 days the majority ofthe radioactivity had been transported down the axon. In-terestingly, he could detect radioactivity up to 250 mmfrom the injection site only 14 hours after the injection,which corresponds to the product being transportedthrough the axon at 500 mm/day. The majority of ra-dioactivity traveled the previously observed rate of only

▲ FIGURE 20.1 Fast axonal transport was characterized by

observing the movement of radioactively labeled proteins

along the length of the axon. Researchers injected [3H]leucineinto dorsal root ganglia of animals. The radioactively labeledleucine is incorporated into proteins, which are subsequentlytransported within the axon. At various time points after injec-tion, the nerves are removed and the axons sectioned and ana-lyzed for the presence of radioactivity. The figure shows distribu-tion of radioactivity throughout the axon at 2 hours (drawn inblue) and 8 hours (drawn in purple) after injection. [Adapted fromS. Ochs et al., 1969, Science 163:686.]

3060 0 30 60 90 120 150

102

103

104

105

106

101

Dorsal root Nerve

mm

2 hrs

8 hrs

Injection

1.3 mm/day, suggesting that more than one mechanismtransported some proteins.

Working with a similar system, Ochs further charac-terized this fast component of axonal transport. While herepeated the experiments Lasek had reported, he lookedat transport using shorter time points after injection. Ochssectioned nerves from 2 to 8 hours after injection (see Fig-ure 19.1), which allowed a more complete characteriza-tion of fast axonal transport. As Lasek had observed, themajority of the radioactivity remained in the ganglia dur-ing these short time points. A small portion, however, trav-eled rapidly within the axon. After 2 hours, Ochs coulddetect radioactivity more than 90 mm away from the in-jection site; by 8 hours, he observed radioactivity up to150 mm from the injection site. From these data, he esti-mated the rate of fast axonal transport to be 410 mm/day.

DiscussionThrough a series of carefully controlled experiments,Lasek and Ochs were able to demonstrate that some pro-teins are transported within the neurons at much fasterrates than others are. It is now known that biomoleculesand organelle travel in the axon at three different rates.The rapidly transported proteins that Lasek and Ochs ob-served were likely part of vesicles or the smooth endo-plasmic reticulum that are now known to move by fastaxonal transport. These vesicles carry neurotransmittersto the axon terminus. It has been shown that these vesi-cles are carried along microtubules in a process that re-quires adenosine triphosphate (ATP). Subsequently, scien-tists have shown that families of molecular motor proteins,the dyneins and the kinesins, power the movement of vesi-cles by fast axonal transport. The study of movementalong the axon—much of which is grounded in Lasek and Ochs’s initial observations of fast axonal transport—remains an exciting field for researchers.


BackgroundThe question of how an organism develops from a fertil-ized egg continues to drive a large body of scientific re-search. Whereas such research was classically the concernof embryologists, the developing understanding of geneexpression in the 1980s brought new approaches to an-swer this question. One such approach was to examinethe pattern of gene expression in both the oocyte and thenewly fertilized egg. Ruderman and Hunt were among thebiologists who took this approach to the study of earlydevelopment.

Biologists had well characterized the early develop-ment of a number of marine invertebrate systems. Dur-ing the early stages of development, the embryonic cellsgrow synchronously, which allows an entire populationof cells to be studied at the same stage of the cell cycle.Researchers had established that a large portion of themRNA in the unfertilized oocyte is not translated. Uponfertilization, these maternal mRNA are rapidly translated.Previous studies had shown that when fertilized eggs aretreated with drugs that inhibit protein synthesis, cell di-vision could not take place. This suggested that the ini-tial burst of protein synthesis from the maternal mRNAis required at the earliest stages of development. Ruder-

CELL BIOLOGY EMERGING FROM THE SEA: THE DISCOVERY OF CYCLINS

From the first cell divisions after fertilization to aberrant divisions that occur in

cancers, biologists have long been interested in the life cycle of the cell. The

life of a dividing cell has been divided into stages known collectively as the cell

cycle. While studying early development in marine invertebrates in the early

1980s, Joan Ruderman and Tim Hunt discovered the cyclins, which are important

regulators of the cell cycle.


man and Hunt, while teaching a physiology course at theMarine Biological Lab, began a set of experiments de-signed to uncover the genes that were expressed at thispoint as well as the mechanism by which this burst of pro-tein synthesis was controlled.

The ExperimentIn a collaborative project, Ruderman and Hunt looked atregulation of gene expression in the fertilized egg of thesurf clam Spisula solidissima. Whereas it was known thatoverall protein synthesis rapidly increased upon fertiliza-tion, they wanted to find out whether the proteins ex-pressed in the earliest stage of development, the two-cellembryo, were different from those expressed in the un-fertilized egg. When either oocytes or two-cell clam em-bryos are treated with radioactively labeled amino acids,the cell takes up the amino acids, which are subsequentlyincorporated into newly synthesized proteins. Using thistechnique, Ruderman and Hunt monitored the pattern ofprotein synthesis by breaking open the cells, separating theproteins using SDS-polyacrylamide gel electrophoresis(SDS-PAGE), then visualizing the radioactively labeledproteins by autoradiography. When they compared the


pattern of protein synthesis in the oocyte to that in thetwo-cell embryo, they saw that three different proteins thatwere either not expressed or expressed at an extremelylow-level in the oocyte were highly expressed in the em-bryo. In a subsequent study, Ruderman examined the pat-tern of protein expression in the oocytes of the starfishAsterias forbesi as they mature. She again observed the in-creased expression of three proteins of similar size to thosethat she and Hunt had seen in surf clam embryos.

Soon afterward, in a third study, Hunt examined thechanges in protein expression during the maturation andfertilization of sea urchin oocytes. This time he performedthe experiment in a slightly different manner. Rather thantreating the oocytes and embryo with radioactively labeledamino acids for a set time period, he labeled the cells con-tinuously for more than 2 hours, removing samples foranalysis at 10-minute intervals. Now, he could monitorthe changes in protein expression throughout the earlystages of development. As had been shown in other or-ganisms, the pattern of protein synthesis was altered whenthe sea urchin oocyte was fertilized. Three proteins—rep-resented by three prominent bands on an autoradio-graph—were expressed in the embryos, but not in theoocytes. Interestingly, the intensity of one of these bandschanged over time; the band was intense at the early timepoints, then barely visible after 85 minutes. It increased inintensity again between 95 and 105 minutes. The inten-sity of the band, representing the amount of the proteinin the cell, appeared to be oscillating over time. This sug-gested that the protein had been quickly degraded and thensynthesized again.

Because the time frame of the experiment coincidedwith early embryonic cell divisions, Hunt next askedwhether the synthesis and destruction of the protein wascorrelated with progression of the cell cycle. He examineda portion of cells from each time point under a micro-scope, counting the number of cells dividing at each timepoint where samples had been taken for protein analysis.Hunt then correlated the amount of the protein present inthe cell with the proportion of cells dividing at each timepoint. He noticed that the level of expression of one ofthe proteins was highest before the cell divided and low-est upon cell division (see Figure 13.1), suggesting a cor-relation with the stage of the cell cycle. When the sameexperiment was performed in the surf clam, Hunt saw thattwo of the proteins that he and Ruderman had describedpreviously displayed the same pattern of synthesis and de-struction. Hunt called these proteins cyclins to reflect theirchanging expression throughout the cell cycle.

1 hr 2 hrs

25

50

75

Cle

avag

e in

dex

Protein B

Cyclin

▲ FIGURE 21.1 This figure compares the changing levels of

sea urchin cyclin (drawn in blue) to a control protein (drawn

in purple) throughout the progression of the cell cycle. Theoverall level of cyclin increases over time, and then it is rapidlydestroyed as the cells approach division. This pattern appears torepeat through each cell division. Meanwhile, the overall level ofthe control protein continues to increase throughout the time pe-riod of the experiment. [Adapted from T. Evans et al., 1983, Cell33:391.]

DiscussionThe discovery of the cyclins heralded an explosion of in-vestigation into the cell cycle. It is now known that theseproteins regulate the cell cycle by associating with cyclin-dependent kinases, which, in turn, regulate the activitiesof a variety of transcription factors that direct progressionthrough the cell cycle. As with so many key regulators ofthe cellular functions, it was soon shown that the cyclinsdiscovered in sea urchins and surf clams are conserved ineukaryotes from yeast to man. Since the identification ofthe first cyclins, scientists have identified at least 15 othercyclins that regulate all phases of the cell cycle.

In addition to the basic research interest in these pro-teins, the cyclins’ central role in cell division has madethem a focal point in cancer research. Cyclins are involvedin the regulation of several genes that are known to playprominent roles in tumor development. Scientists haveshown that at least one cyclin, cyclin D1, is overexpressedin a number of tumors. The role of these proteins in bothnormal and aberrant cell division continues to be an ac-tive and exciting area of research today.


BackgroundThe proper development of an embryo from a fertilized egghas long posed a fascinating and difficult question. To ex-amine complex processes, biologists often use genetic ap-proaches, which involve collecting mutant organisms thatdiffer from the normal or “wild-type” organism. In study-ing developmental biology, a geneticist’s approach involveslooking for mutant organisms that display an obvious de-fect in overall formation. Early work uncovered a numberof genes involved in the development of the fruit flyDrosophila melanogaster. In the first genes examined, mu-tations resulted in the birth of flies with obvious physicaldefects, such as the presence of an extra set of wings. Be-cause this approach relied on examining viable flies withphysical malformations, it missed many developmentallyimportant genes that, when mutated, result in the death ofthe fly embryo.

In the late 1970s, Nüsslein-Volhard and Wieschaus be-gan their pioneering work on the development ofDrosophila embryos. They sought to identify as many genesin the developmental process as possible by looking forgenes that resulted in the death of the embryonic fly. Theirwork unveiled several key genes active in the early devel-opment of not only Drosophila, but higher organisms aswell.

The ExperimentGeneticists develop systematic methods, known as geneticscreens, to search for mutations that affect biologicalprocesses. Nüsslein-Volhard and Wieschaus had to considerseveral previous observations on Drosophila developmentwhen they designed their screen. First, they knew that genesexpressed in the egg, called maternal-effect genes, as wellas genes expressed after fertilization in the developing em-bryo, called zygotically active genes, controlled the early de-velopment of an embryo. They chose to focus on isolatingzygotically active mutants. Second, they had to consider thatthe Drosophila genome is diploid, which means that theprogeny receives a copy of each gene from both parents.Scientists had previously demonstrated that Drosophila re-quired only a single wild-type copy of most genes in orderto develop into a viable fly. This made it likely that the de-velopmentally active mutants that the screen was lookingfor would be recessive. Therefore, to see defects resultingfrom mutations in these genes required breeding the mutantDrosophila such that it was homozygous for the mutations.

The overall mutation rate in a naturally occurring pop-ulation is quite low. If a geneticist were to search for mu-tants in a natural population, he or she would have to ex-amine a large number of individuals. To circumvent thisdifficulty, Nüsslein-Volhard and Wieschaus induced muta-

USING LETHAL MUTATION TO STUDY DEVELOPMENT

One of the most fascinating questions in developmental biology concerns the

proper formation of an embryo. How does a fertilized egg “know” how to

form a complex organism? Scientists have puzzled over how to address this ques-

tion for a long time. In 1980, Christiane Nüsslein-Volhard and Eric Wieschaus

used the fruit fly Drosophila melanogaster to demonstrate how a genetic

approach could be used to address this problem.



tions in a population at the onset of the screen, then cre-ated inbred lines to assure that each fly they examinedwould carry the induced mutation on each chromosome.They fed a mutagenic chemical to male flies, then matedthem to a genetically defined population of female flies ina process known as a genetic cross. The resulting progenywould be heterozygotes because they would have the mu-tation only on the chromosome they received from the fa-ther. To assure the homogeneity of the genetic background,the heterozygote males were mated again to females of thesame genetic background, establishing an inbred line. Fi-nally, males and females from the inbred line were matedto each other, and the progeny were examined for the de-sired phenotype, embryonic death.

Using this screen, Nüsslein-Volhard and Wieschausamassed a large collection of mutants. The next step wasto assign the mutant Drosophila to specific classes, basedon their phenotype. They focused on the segmentation ofthe larvae. Whereas all mutants in this screen necessarilydisplayed the phenotype of embryonic lethality, they dif-fered greatly in their segmentation defects. To classify thesedefects, Nüsslein-Volhard and Wieschaus examined the lar-

vae under the microscope. They compared the body patternof a wild-type larva (see Figure 14.1, far left), which is vi-able, to those of the embryonic lethal mutants. By com-paring these patterns, they uncovered three classes of genesthat affect segmentation, which they called segment polar-ity, pair-rule, and gap.

Gap mutants are missing up to eight segments from theoverall body, without regard to symmetry (see Figure 14.1),which results in a smaller body type. Three mutants—knirps, hunchback, and the previously characterized Krüp-pel—fell into this class. The next class of mutants, the seg-ment polarity mutants, has the same overall number of seg-ments as the wild-type larvae. The mutation resulted in adeletion of the body pattern within a segment. The deletedsegment was replaced by a mirror image of the portion thatremained. Nüsslein-Volhard and Wieschaus’s initial screenuncovered six mutants of this class, three of which, goose-berry, hedgehog, and patch, had not been previously ob-served. The final set of mutations, the pair-rule mutation,resulted in deletion of alternating segments of the body,which caused a shorter body formation. Five previously un-characterized mutants, paired, even-skipped, odd-skipped,

Normal Krüppel hunchback knirps

▲ FIGURE 22.1 Examples of three embryonic lethal muta-

tions uncovered in Christiane Nüsslein-Volhard and Eric

Wiecshaus’s screen for segmentation mutants. The pheno-type of a viable embryo is shown on the left (Normal). Thephenotypes of three mutant embryos from the gap class, Krüp-

pel, hunchback, and knirps, also are shown. Thoracic segmentsare labeled T1–T3, whereas abdominal segments are designatedA1–A8. Gap mutants are missing entire segments from the bodyplan, as illustrated by the labeled segments on the left. [From C.Nüsslein-Volhard and E. Wieschaus, 1980, Nature 287:795.]


barrel, and runt, as well as one known mutant, engrailed,were placed in this class.

DiscussionBy the first report of their screen, Nüsslein-Volhard and Wieschaus had identified 15 mutants that affected segmen-tation. Of these, only five were previously identified genes.When they completed the study—often referred to as theHeidelberg screens—they had identified 139 different genesthat, when mutated, resulted in embryonic death. These mu-tations fell into 17 different classes. These mutants formedthe base for the past 20 years of research into the develop-

ment of Drosophila. As molecular techniques evolved, sci-entists cloned many of these genes and characterized theirgene products.

The majority of proteins encoded by the genes have beenshown to be transcription factors, but the screen also un-covered signaling molecules, receptors, enzymes, adhesionmolecules, cytoskeleton proteins, and proteins whose func-tions remain unknown. Scientists interested in mammaliandevelopment have studied homologues of the Drosophilagenes uncovered by Nüsslein-Volhard and Wieschaus, andhave shown them to be important in mammalian develop-ment as well. In 1995, the Nobel Foundation awarded itsprize for Physiology and Medicine to Nüsslein-Volhard andWieschaus for their pioneering work.


BackgroundDevelopmental biologists have long noted that some cellsdie during the normal development of a multicellular or-ganism. This process—called apoptosis or programmedcell death—remained a puzzling phenomenon for manyyears. Early research in the field concentrated on identi-fying cells that were fated to die. Until the middle of the1980s, scientists knew little about the mechanism by whichthe cell controlled this process. At this time, Horvitz be-gan his investigations into the genetics of programmed celldeath in the nematode Caenorhabditis elegans (C. elegans).

C. elegans is a powerful model system for studying thegenetics of complex developmental processes. It is a smallmulticellular organism, made up of just more than 1,000cells, which allowed scientists to trace the developmental lineage of each cell of the organism. Previous studies haddefined precisely which cells in C. elegans were fated to die during development. When examined using Nomarskidifferential contrast microscopy, cells fated to die becamehighly refractile for a few minutes before cell death oc-curred. In the late 1970s, researchers isolated two celldeath mutants of C. elegans: ced-1 and ced-2. These mu-tants extend the life of dying cell, causing it to remain re-fractile for hours rather than minutes (Figure 23.1).

HUNTING DOWN GENES INVOLVED IN CELL DEATH

During the development of multicellular organisms, certain cells are destined

to die. Scientists have directed much research toward understanding this

process of programmed cell death. Some sought to understand why a cell would

be destined to die during development, whereas others asked how a cell regulates

this form of death. In 1986, H. Robert Horvitz provided clues by examining the

genetics of cell death using a well-characterized model system, the nematode

Caenorhabditis elegans.


Horvitz used these mutants to look for more genes in-volved in the control of programmed cell death.

The ExperimentGeneticists analyze complex processes by looking for or-ganisms that display traits or phenotypes that result froman alteration in normal function of a gene. To perform agenetic analysis of programmed cell death, a geneticistlooks either for cells that escape programmed cell death,or cells that undergo programmed cell death when theyshould survive. Finding such mutant organisms in a nat-urally occurring population would be difficult because theoverall mutation rate of an organism is rather low. To fa-cilitate the search, mutations are induced, often by treat-ing them with mutagenic chemicals. This enriches the pop-ulation for mutants. Because mutagenic chemicals willinduce mutations in all genes, not just those involved inthe process being studied, scientists carefully devise geneticscreens to guide their studies.

To analyze the control of cell death in C. elegans,Horvitz designed a genetic screen for mutations that alterthe process. In the screen described here, he looked formutations that allowed cells that would normally die


during development to survive. A second important partof the screen was the choice of organisms to study. Ratherthan look for mutant progeny of wild-type nematodes,Horvitz looked at the progeny of ced-1 mutants. In ced-1 mutants, cells that die by programmed cell death are notengulfed and immediately eliminated from the organism.This causes them to remain highly refractile under No-marski optics for longer than a cell in a wild-type organ-ism. By using ced-1 mutants in his studies, Horvitz hadan increased time frame to look for mutant nematodes inwhich the cells normally fated to die escape programmedcell death.

(a)

▲ FIGURE 22.1 Screening for C. elegans genes involved in

programmed cell death were observed using Nomarski dif-

ferential contrast microscopy. (a) Newly hatched larva carryinga mutation in the ced-1 gene. Because mutations in this geneprevent engulfment of dead cells, highly refractile dead cells ac-cumulate facilitating their visualization. The arrows indicate threehighly refractile cells. (b) Newly hatched larva with mutations inboth the ced-1 and ced-3 genes. Using the nuclei indicated bythe arrowheads as orientation points, one can compare panels aand b. In panel b, notice that the three highly refractile cells seenin panel a are not observed. The absence of refractile dead cellsin these double mutants indicates that no cell deaths occurred.Thus ced–3 was identified as a gene involved in programmedcell death. [From H. M. Ellis and H. R. Horvitz, 1986, Cell 91:818.Courtesy of Hilary Ellis.]

(b)

In his initial screen for genes involved in programmedcell death, Horvitz treated ced-1 mutant nematodes witha mutagenic chemical and allowed them to reproduce fortwo generations, producing a genetically defined line thatcarries mutations on both copies of the chromosome. Be-cause the progeny are homozygous for all mutations, hecould now observe the phenotype of mutations that arerecessive. Once the organisms were bred to homozygos-ity, he analyzed the second generation for mutations thataffected cell death. Specifically, he compared the highly re-fractile dying cells in ced-1 nematodes to the same cells inmutagenized progeny. These cells displayed the same phe-notype in the majority of larvae examine. In a small num-ber of larvae, including those that harbored mutations ingenes that control programmed cell death, these cells donot die, and hence do not become refractile. This initialscreen uncovered several recessive mutants that mappedto a single gene that he called ced-3 (Figure 23.1).

Horvitz went on to characterize the phenotype of theced-3 mutants, taking advantage of the fact that the iden-tity of all cells destined to undergo programmed cell deathwas known. By examining the fate of cells that normallyundergo programmed cell death, he showed that mutationin ced-3 blocked this process completely (see Table 23.1).He then followed these surviving cells through the C. el-egans life cycle. When compared with wild-type nema-todes, ced-3 mutants reproduced normally and showed nobehavioral abnormalities. The primary difference ap-peared to be the extra cells present in ced-3 mutants dueto the absence of programmed cell death. Horvitz pro-ceeded to analyze a number of mutations within the ced-3 gene, known as different alleles of the gene. Each alleleresulted in the identical phenotype, survival of cells thatshould be destined to die. This suggested to Horvitz thatmutations in ced-3 resulted in the loss or decreased ex-pression of an essential gene in the programmed cell deathpathway. He had isolated the first gene required for con-trol of programmed cell death in C. elegans.

TABLE 22-1 Mutations in Ced-3 Eliminates Cell Death

Genotype Average Number of Cell Deaths Observed

First larval stage Postembryonic

Wild type ND 13ced-1 28 11.23ced-3 0.3 0.04

ND = Not determined[Data adapted from H. M. Ellis and H. R. Horvitz, 1986, Cell44:819.]


DiscussionThe isolation of the ced-3 mutant was merely the first stepin Horvitz’s efforts to dissect the genetics of programmedcell death in C. elegans. In addition to ced-3, he uncov-ered two other essential genes in the pathway, as well as10 other genes that are involved in this process. Thesegenes control all aspects of the process of cell death, fromthe initial decision to die, to the killing, engulfment, anddegradation of the dead cell. As is often seen, these genesthat control cell death in C. elegans have counterparts inhigher organisms, including humans. The importance ofthese genes in the regulation of cell growth in humans hasbecome apparent. The human homologues of two genesisolated by Horvitz have been implicated in cancer.


BackgroundUnderstanding the molecular and cellular events that oc-cur in cancer has long been a goal of biological research.Scientists have sought to understand how a normal cellbecomes transformed into a tumor cell. Although they rec-ognized early in the century that some viruses could in-duce tumors in animals, it wasn’t until many years laterthat they investigated the viral causes of cancer in depth.Such investigation was aided by the development of tech-niques to study viruses in cultured cells rather than in liv-ing organisms. In the late 1950s, Dulbecco adapted a num-ber of techniques used to manipulate bacteriophages—viruses that infect prokaryotic cells—for use in studyinganimal viruses in cultured eukaryotic cells. He then turnedhis attention to studying cell transformation by viruses incultured cells, using the DNA tumor viruses simian virus40 (SV40) and polyoma virus.

Both SV40 and polyoma viruses could infect a num-ber of different types of cells in culture. Cells that are sus-ceptible to infection by these viruses fall into two classes:permissive cells, which produce virus after infection, andnon-permissive cells, which do not. Whereas non-permis-sive cell lines did not produce virus after infection, these

STUDYING THE TRANSFORMATIONOF CELLS BY DNA TUMOR VIRUSES

Not many diseases have spurred scientists to research and understand their

causes more than human cancers have. These scientists recognized long ago

that some viruses could induce tumors in animals, and in the 1960s and 1970s,

research in this field surged. One of the pioneers in the field was Renalto

Dulbecco, who, in 1968, reported that a tumor-causing virus could insert its DNA

into the genome of the cell it transformed.


were the only cells that the tumor viruses could transform.Something was happening in the non-permissive cell linesthat caused them to be transformed, rather than producevirus. Dulbecco noted that a similar phenomenon had beenobserved in bacteriophage infections. Some phages causethe rapid production of new phages in a lytic infection.Other phages—through the process of lysogeny—lay dormant in the infected cell, while its DNA became inte-grated into the bacterial genome. Dulbecco wondered ifviral infection in non-permissive cells might be similar tolysogenous phage infection in bacteria. He and othersdemonstrated that the viral DNA could be detected in cellstransformed by polyoma or SV40 viruses. Could this vi-ral DNA be integrated into the cellular DNA? In the late1960s, Dulbecco set out to find the answer.

The ExperimentTo determine the state of SV40 DNA in transformed cells,Dulbecco used nucleic acid hybridization, which is a pow-erful technique that can detect a relatively small DNA se-quence within a larger sample of DNA. In his experiment,Dulbecco isolated viral DNA from purified SV40 parti-


cles. He then used purified RNA polymerase to transcribethe viral DNA into RNA in vitro. He labeled the RNA ra-dioactively by including [3H]cytosine triphosphate (CTP)in the in vitro transcription reaction, creating a radioac-tively labeled probe. Next, he prepared genomic DNAfrom SV40-transformed cells, heated it to separate thedouble-stranded DNA into two single-stranded molecules,and then bound single-stranded DNA to nitrocellulose fil-ters. He then treated the single stranded DNA bound tothe filters with the radioactively labeled RNA probe.Through base-pairing interactions, the radioactively la-beled RNA specifically hybridized to the SV40 DNA. Af-ter washing the filters to remove unhybridized RNA, heanalyzed the filters by scintillation counting. Under opti-mal conditions, radioactivity on the membranes correlateswith the presence of SV40 DNA in the sample. Indeed,Dulbecco successfully employed this technique to detectSV40 DNA in the transformed cells.

Once he determined that the viral DNA was presentin the transformed cells, Dulbecco proceeded to determinethe form and the location of SV40 DNA in a transformedcell. To do this, he took advantage of physical differencesbetween the SV40 viral DNA and the genomic DNA iso-lated from the infected 3T3 cells (SV3T3). SV40 DNA iso-lated from viruses is in a circular supercoiled form, whichcan be separated from SV3T3 genomic DNA by equilib-rium density centrifugation through a gradient of cesiumchloride (CsCl) in the presence of ethidium bromide.Ethidium bromide intercalates into linear DNA more read-ily than circular DNA, changing its density and thus al-lowing him to separate supercoiled viral DNA from lin-ear genomic DNA. As a control, Dulbecco performed thesame analysis on polyoma virus–transformed 3T3 cells(Py3T3). He immobilized DNA isolated by these proce-dures onto nitrocellulose filters and hybridized the ra-dioactively labeled SV40 RNA. He found that the SV40RNA hybridized only to the linear fraction of DNA fromthe SV3T3 cells (see Table 24.1A). The level of hy-bridization to the supercoiled DNA was no greater in theSV3T3 cells than it was in the Py3T3 cells, indicating thatany radioactivity detected in this fraction was at back-ground levels. As an additional control, Dulbecco addedsupercoiled DNA isolated from SV40 to the Py3T3 cellextract. In these cells, he could detect the DNA specifi-cally in the supercoiled DNA fraction of the DNA isolatedfrom these Py3T3 cells, and not in the linear DNA frac-tion (see Table 24.1A). From these experiments, he con-cluded that the SV40 DNA was not in its supercoiled, viralform in transformed cells.

To assure that the SV40 DNA in the linear fractionwas part of the SV3T3 genomic DNA, and not linearizedSV40 viral DNA, Dulbecco performed alkaline sucrosegradients. He layered the cells onto the alkaline gradient,then incubated them to allow the cells to break open. This

procedure minimized inadvertent mechanical shearing ofcellular DNA. Once lysis was complete, the cellular DNAwas sedimented in the sucrose gradient. Dulbecco thencompared the hybridization of SV40 RNA to DNA iso-lated from SV3T3 and Py3T3 and found that it specifi-cally hybridized to the SV3T3 cells (see Table 24.1B). Be-cause the SV40 DNA always hybridized to the highmolecular weight genomic DNA, he concluded that it wascovalently attached to the cellular DNA.

DiscussionDulbecco’s demonstration that the SV40 DNA was inte-grated into the genome of transformed cells began a newwave of thinking about cellular transformation. With theadvent of restriction endonucleases, as well as other mol-ecular biology techniques, scientists demonstrated thatSV40 integrates into random sites in the host cell genome.Researchers have found that a number of other tumor-inducing viruses, including the RNA-based retroviruses, integrate into the host cell genome. They later showed thatviral integration could disregulate the expression of keygenes in cell growth, which would contribute to tumorformation.

By inspecting the sites of viral insertion, scientists havediscovered a variety of oncogenes. These scientists predi-cated this work on Dulbecco’s initial studies on DNA tu-mor viruses and their ability to integrate into the hostgenome. In 1975, the Nobel Foundation awarded its prizefor Physiology and Medicine to Dulbecco for his vast con-tributions to this field.

TABLE 23-1 Demonstration that SV40 DNA IsIntegrated in Transformed Cells

A. CSCL CENTRIFUGATION

Cells Circular DNA(CPM) Linear DNA (CPM)

SV3T3 69 � 5 358 � 2Py3T3 64 � 7 129 � 12Py3T3 � SV40

viral DNA 385 � 11 198 � 5

B. ALKALINE SUCROSE GRADIENTS

Cells CPM hybridized

SV3T3 620 � 12Py3T3 248 � 2

CPM � Counts per minute.[Data adapted from Sambrook et al., Proc. Natl. Acad. Sci USA,1968, 59:1290 and 1294.]


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