Classic Experiments

7/29/2019 Classic Experiments

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3.1

Bringing an EnzymeBack to Life

By the 1950s, scientists realized that DNA held the code that allowedproteins to be synthesized. Nevertheless, how a chain of aminoacids 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 thatthen fold into complex structures. The unfolded amino acidchain 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 formhas long been a mystery. Does the protein fold correctly asit is synthesized or does it require the action of other pro-teins to correctly fold it? Can it correctly fold on its ownspontaneously?

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 ofthe major anchors holding together the structure of secretedproteins. He believed that the protein itself contained all theinformation necessary for proper protein folding. He pro-posed the thermodynamic hypothesis, which stated thatthe biologically active structure of a protein was also themost thermodynamically stable under in vivo conditions. Inother words, if the intracellular conditions could be mimic-

ked in a test tube, then a protein would naturally fold inits active conformation. He began his work on a secreteenzyme, bovine pancreatic ribonuclease, and studied its abity to properly fold outside of the cell.

The ExperimentProteins perform a wide variety of functions in the cell. Rgardless of its function, a protein must be properly foldeto carry out its biological role. For protein folding studiit is best to study an enzyme whose biological activity cabe easily monitored by performing in vitro. Anfinsen choa small, secreted protein, the enzyme ribonuclease, in whiche could monitor proper folding by assaying its ability

catalyze the cleavage of RNA.Ribonuclease, a secreted protein, is active under oxidi

ing conditions in vitro. The tertiary structure of active rbonuclease is held together by four disulfide bridges. Addina reducing agent, which reduces the disulfide bond betweetwo cysteine side chains to two free sulfhydryl groups, cadisrupt this covalent interaction. Complete denaturation ribonuclease requires treatment with a reducing agenAnfinsen monitored the reduction of ribonuclease by mesuring the number of free sulfhydryl groups present in th

Classic Experiment


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TABLE 3-1 Cell-free Refolding of Ribonuclease

Activity as a Percent ofConcentration 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 ofBiological Chemistry 236:1362.]

protein. In the oxidized state, there are no free sulfhydrylgroups 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 freesulfhydryl 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 firstdenature the protein. Proteins are easily denatured by heat,mechanical disruption such as shaking, and chemical treat-ment. Proteins with disulfide bridges require an additionalmeasure of treatment with a reducing agent to break apartthese covalent bonds. To denature ribonuclease, Anfinsenfirst reduced the disulfide bridges with thioglycolic acid. Hethen denatured the protein by using a high concentration ofurea and incubating the solution at room temperature. Hedemonstrated that this treatment rendered the enzyme in-active by showing that ribonuclease was now unable to cat-alyze the cleavage of RNA. Using the spectrophotometricassay, he went on to show that the inactive ribonuclease con-

tained eight sulfhydryl groups, which corresponded to thefour broken disulfide bridges. With a completely reduced,denatured protein in hand, Anfinsen then could ask: Can adenatured enzyme correctly fold in vitro and become activeagain?

To find the answer, Anfinsen allowed a solution of re-duced, denatured ribonuclease to oxidize. He removed theurea from the denatured enzyme by precipitation. Next, heresuspended the urea-free denatured ribonuclease in abuffered solution and incubated it for two to three days. Ex-posure to molecular oxygen in the atmosphere oxidized thecysteine residues. He then compared the activity of this re-natured ribonuclease to that of the native enzyme. In initial

experiments, 1219 percent of the previously inactive pro-tein were able to catalyze the cleavage of RNA once again.Proteins aggregate at high concentrations, which makes itdifficult for them to fold properly. By decreasing the over-all concentration of ribonuclease in solution, Anfinsenshowed 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 thatthe information for the protein folding is contained in theprotein itself.

DiscussionThrough careful experiments, Anfinsen demonstrated thatthe information required to properly fold a protein is con-tained in its primary sequence. His careful analysis of thechemistry of this process answered a fundamental questionin biology. He went on to demonstrate the cell-free refold-ing of other enzymes, including proteins lacking disulfidebridges. While it is possible to properly fold a number ofproteins outside of the normal protein-processing machin-ery in the cell, this process is greatly accelerated in vivo bya number of enzymes. Anfinsen continued to study theprotein-folding problem. Although the thermodynamic

hypothesis does not hold true for all proteins, Anfinsensdemonstration of the cell-free refolding of ribonuclease madea mark on the field of biochemistry. In 1972, he receivedthe Nobel Prize for Chemistry for his work.


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Background

Proteins are made from combinations of 20 differentamino acids. The genes that encode proteinsthat is, spec-ify the type and linear order of their component aminoacidsare located in DNA, a polymer made up of onlyfour different nucleotides. The DNA code is transcribedinto RNA, which is also composed of four nucleotides.Nirenbergs 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 proteinsynthesis.

At the outset of his studies, much was already known

about 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 aminacids, beginning protein synthesis. The nascent protechain is elongated by the subsequent binding of additiontRNAs and formation of a peptide bond between th

incoming amino acid and the end of the growing chaiAlthough this general process was understood, the quetion remained: How does the mRNA direct protesynthesis?

When attempting to address complex processes such protein synthesis, scientists divide large questions into series of smaller, more easily addressed questions. Prior Nirenbergs study, it had been shown that when phenyalanie charged tRNA was incubated with ribosomes anpolyuridylic acid (polyU), peptides consisting of onphenylalanine were produced. This finding suggested ththe mRNA codeword, or codon, for phenylalanine

made up of the nucleosides containing the base uracSimilar studies with polycytadylic acid (polyrC) anpolyadenylic acid (polyrA) showed these nucleosides cotaining the bases cytadine and adenine made up thcodons for proline and lysine, respectively. With thknowledge in hand, Nirenberg asked the question: Whis the minimum chain length required for tRNA binding ribosomes? The system he developed to answer this quetion would give him the means to determine which aminacylated tRNA would bind which m-RNA codon, effetively cracking the genetic code.


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The Experiment

The 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 tRNArecognition.

Before he could begin his experiment, Nirenberg neededboth a means to separate the complex from unboundcomponents 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

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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

Phe

Trinucleotide 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

ArgArg

Phe

Leu

Assay developed by Marshall Nirenberg and his collaborators for deciphering the genetic code. They prepared 20 E. coliextracts 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 a

nylon filter without binding (left panel); ribosomes, however, bind to the filter (center panel). Each of the 64 possible trinucleotides was

tested separately for its ability to attract a specific tRNA by adding it with ribosomes to different extract samples. Each sample was then

filtered. If the added trinucleotide causes the radiolabeled aminoacyl-tRNA to bind to the ribosome, then radioactivity is detected on the

filter; otherwise, the label passes through the filter (right panel). By synthesizing and testing all possible trinucleotides, the researchers

were 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, Science145:1399].


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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!

Discussion

Combined with the technology to generate trinucleotidof known sequence, Nirenbergs assay provided a way assign each specific amino acid to one or more speciftrinucleotides. Within a few years, the genetic code wacracked, all 20 amino acids were assigned at least one trnucleotide, and 61 of the 64 trinucleotides were found tcorrespond to an amino acid. The final three trinucleotides, now known as stop codons, signal termintion of protein synthesis.

With the genetic code cracked, biologists could reathe gene in the same manner that the cell did. Simply bknowing the DNA sequence of a gene, scientists can nopredict the amino acid sequence of the protein it encodeFor his innovative work, Nirenberg was awarded thNobel Prize for Physiology and Medicine in 1968.


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Background

In 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 viruss 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 genomic

RNA, 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 indepedently prove Temins model.

The Experiment

Remarkably, these two scientists traveled separate patways to the same critical set of experiments. Both begawith pure stocks of virus, which they then partially dirupted using nonionic detergents. With a stock odisrupted viruses in hand they could ask the critical quetion: Can a retrovirus perform DNA synthesis? To answthis question, each groups added radiolabeledeoxythymidine triphosphate (dTTP) along with the oththree deoxynucleotide triphosphates (dATP, dCTP, dGTto the virion preparations, and looked for the incorpor

tion of radioactive dTTP into DNA. Indeed, in each expeiment radiolabeled dTTP was incorporated into nucleacid. When Baltimore added a radiolabeled ribonucleotidtriphosphate (rNTP) and the three other ribonucleotidtriphosphates to disrupted viruses, he could detect no RNsynthesis. To prove that the product being formed was fact DNA, they treated it with enzymes that specificaldegrade either RNA (ribonuclease, or RNase) or DN(deoxyribonuclease, or DNase). They found the product be sensitive only to DNase. The results of these simpexperiments, summarized in the Table, showed that a

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.



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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 to

believe that reverse transcriptase existed because its activ-ity violated the central dogma. Subsequent isolation andcharacterization of the paradigm-shattering enzyme soonconvinced the skeptics.

Discussion

Temin 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 himto 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 Temins and Baltimores 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 dpm

Virions 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.


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Background

During 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 DNAs 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 contains

one 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 wenow available for a definitive test to determine whethDNA replication occurs by a semiconservative or conserative mechanism.

The Experiment

Meselson and Stahl reasoned that if one could label thparental DNA in such a way that it could be distinguishefrom the daughter DNA, the replication mechanisms coube distinguished. If DNA replication is semiconservativthen after a single round of replication, all DNA moleculshould be hybrids of parental and daughter DNA strandIf replication is conservative, then after a single round replication, half of the DNA molecules should be compose

only of parental strands and half of daughter strands.To differentiate parental DNA from daughter DNAMesselson and Stahl used heavy nitrogen (15N). Thisotope contains an extra neutron in its nucleus, giving a higher atomic mass than the more abundant lighnitrogen (14N). Since nitrogen atoms make up part of thpurine and pyrimidine bases in DNA, it was easy to labE. coli DNA with 15N by growing bacteria in a mediucontaining 15N ammonium salts as the sole nitrogesource. After several generations of growth, the bactercontained only 15N-labeled DNA. Now that the parent

PROVING THAT DNA REPLICATION IS

SEMICONSERVATIVE

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. In1958, Mathew Meselson and Franklin Stahl used the newly developed techniques

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

semiconservative fashion.



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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 (

14

N) 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 of14N-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.

Discussion

For 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.


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Oldstrand

Parent strandssynthesizedin 15N

First doublingin 14N

Second doublingin 14N

One band:HH

Two bands:HH + LL

New strands

One bandHH

One band:HL (hybrid)

Two bands:HL + LL

Two bands:HH + LL

Light(14N)

Heavy(15N)

HH

H 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

HL

LL HL

Experimental demonstration by Messelson and Stahl that DNA replication is semiconservative. After several generations of growth in a

medium containing heavy (15N) nitrogen, E. coliwere transferred to a medium containing the normal light isotope (14N). Samples

were removed from the cultures periodically and analyzed by equilibrium density-gradient centrifugation in CsCl to separate heavy-heav

(H-H), light-light (L-L), and heavy-light (H-L) duplexes into distinct bands. The actual banding patterns observed were consistent with the

semiconservative 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. Natl. Acad. Sci. USA 44:671; photographs courtsey of M. Messelson.]


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Background

Eukaryotic 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 organelles 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 homogeneityand 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 ceArmed with these hypotheses and the powerful tool of cetrifugation, de Duve further subdivided the mitochondriarich fraction. First, he identified the light mitochondrifraction, which is made up of hydrolytic enzymes that anow known to compose the lysosome. Then, in a series experiments described here, he identified another discresubcellular fraction, which he called the perioxisomwithin the mitochondrial-rich fraction.

The Experiment

de Duve studied the distribution of enzymes in rat livcells. Highly active in energy metabolism, the liver co

tains a number of useful enzymes to study. To look for thpresence of various enzymes during the fractionation, hrelied on known tests, called enzyme assays, for enzymactivity. To retain maximum enzyme activity, he had take precautions, which included performing all fractioation steps at 0C because heat denatures protein thwould compromise enzyme activity.

de Duve used rate-zonal centrifugation to separate celular components by successive centrifugation steps. Hremoved the rats liver and broke it apart by homognization. The crude preparation of homogenized cells w


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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. The

light 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

In

creasingdensityof

sucrose(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

Relativeconcentration

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 the

organelles 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, and

then enzyme assays were performed. The relative concentration

of active enzyme is plotted on the y-axis; the height in the tube is

plotted on the x-axis. The peak activities of cytochrome oxidase

(top) and acid phosphatase (bottom) are observed near the top of

tube. The peak activity of uricase (middle) migrates to the bottom

of the tube. [Adapted from Beaufay et al., 1964, Biochem J.

92:191.]


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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.

Discussion

de Duves 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 inventory of enzymes in a given cell fraction gave him clues tits function. His careful work resulted in the uncovering two organelles: the lysosome and the perioxisome. His woalso provided important clues to the organelles functioThe lysosome, where de Duve found so many potentialdestructive enzymes, is now known to be an important si

for degradation of biomolecules. The perioxisome has beeshown to be the site of fatty acid and amino acid oxidtion, reactions that produce a large amount of hydrogeperoxide. In 1974, de Duve received the Nobel Prize foPhysiology and Medicine in recognition of his pioneerinwork.


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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 scientistMasatoshi 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 Experiment

In 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 the

new conditions.Puzzled by his difficulty in repeating this seemingly

basic 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

Background

In 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, they

separate 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


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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 cell

adhesion in this cell line.To overcome this problem, Takeichi began studying

cell 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 the

presence of Ca

2

.

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 that

fibroblasts do not form adhesive interactions in culture. Notice how the cells seem to overlap one another. (b) Light micrograph showing

fibroblasts expressing E-cadherin in culture. These cells adhere to one another, as demonstrated by the easily definable boundaries

between 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 cell

surfaces, suggesting that E-cadherin is in fact mediating cell adhesion. (Nagafuchi, A., et al. [1987]. Nature329: 341343.)

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 lung

cells. 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


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

Discussion

Over 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 from

what 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 the

experiment 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.

Takeichis 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 the

gene 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

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


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Background

During 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 move

across the membrane against their concentration gradients.This energy-requiring process was termed active transport.At the time of Skous experiments, the mechanism of

active 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/K1ATPase, a molecular pump that catalyzes active transport.


poses, Skou set out to isolate such an ATPase from a moreadily available source, crab leg neurons. It was durinhis characterization of this enzyme that he discovered thproteins function.

The Experiment

Since the original goal of his study was to characterize thATPase for use in subsequent studies, Skou wanted tknow under what experimental condition its activity wboth robust and reproducible. As often is the case with thcharacterization of a new enzyme, this requires careftitration of the various components of the reaction. Befothis can be done, one must be sure the system is free frooutside sources of contamination.

In order to study the influence of various cationincluding three that are critical for the reactionNaK, and Mg2Skou had to make sure that no contamnating ions were brought into the reaction from anothsource. Therefore, all buffers used in the purification the enzyme were prepared from salts that did not contathese cations. An additional source of contaminatincations was the ATP substrate, which contains three phophate groups, giving it an overall negative charge. Becaustock solutions of ATP often included a cation to balancthe charge, Skou converted the ATP used in his reaction


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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 this

was 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.

Discussion

Skous 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)

g

P

40

30

20

10

0

g

P

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

K350mM/I

K200mM/I

K 120mM/I

K 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 that

increasing K leads to an inhibition of the ATPase activity. The graph on the right shows that with increasing Na, the enzyme activity

increases up to a peak and then levels out. This graph also demonstrates the dependence of the activity on low levels of K. [Adapted

from J. Skou, 1957, Biochem. Biophys. Acta23:394.]


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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 enomous impact on membrane biology, leading to a bettunderstanding of the membrane potential. The generatioand disruption of membrane potential forms the basis many biological processes including neurotransmissioand the coupling of chemical and electrical energy. For thfundamental discovery, Skou was awarded the NobPrize for Chemistry in 1997.


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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.

Mulliss 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 OF

EXPONENTIAL GROWTHTHE

POLYMERASE CHAIN REACTION

I

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

to be realized. Geneticists were uncovering the genetic defects that lead to manyhereditary 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 wadded to the DNA and allowed to anneal to the complmentary region, thereby creating a primer-template complex, one of the substrates for DNA polymerase. Thother substrates, the four deoxynucleotide triphosphat(dNTPs), were then added, so that DNA synthesis couoccur. Although this method was useful for producinradioactively labeled pieces of DNA, it could not amplia DNA sequence, only replicate it.

The Experiment

Mullis designed a method that would actually amplify thamount of target DNA, a prerequisite for detecting a smaDNA sequence within a large complex sample of genomDNA. For instance, the human genome conationsnucleotides of coding sequence. Molecular diagnosis sickle cell anemia requires the detection of one alterenucleotide in one gene amongst the rest of the genome. Taccomplish this, the region of the genome containing thalteration must be amplified.

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

3 1


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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 theDNA 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 of

the 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.

Discussion

Development 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 encodingthe -globingene

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 the

mutation that leads to sickle cell anemia; the other primer is com-

plementary to the () strand and hybridizes upstream of the

mutation. Repeated cycles of DNA denaturation, primer annealing,

and DNA synthesis amplify the target sequences between the

primer-binding sites.


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Background

At the time of Hamilton Smiths work, host restriction wasa well-characterized, yet highly intriguing phenomenon. It

was 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, a

bacterium 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-SPECIFIC

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


non-E.coli DNA, Meselson could not demonstrate ththis cleavage was sequence specific. In fact, proving ththese bacterial enzymes cleave DNA at a specific sequenwould be a tricky manner, as this research was conductebefore the advent of the relatively simple DNA-sequencintechniques now available. Following on Messelsonwork, Smith set out to purify a second restriction enzymthis time from H. influenzae, and to demonstrate that does indeed cleave DNA in a sequence-specific manner.

The Experiment

The first step in the successful purification of a neenzyme is devising an assay that measures the knowactivity of the enzyme as it is being purified. The activi

of a restriction enzyme is to catalyze the cleavage of foeign DNA, so this was the logical activity to monitor. Tdo so, Smith took advantage of the fact that genomDNA from bacteria is quite viscous, however as nucleasbegin to degrade the bacterial DNA, its the overall viscoity decreases. Therefore, Smith could monitor the purifcation of his restriction enzyme by measuring the decreain viscosity of a foreign DNA after treatment with a sample of the protein after each step in the purificatioscheme. Smith mixed cell extracts of H. influenzae wiintact DNA from either H. influenzae or the Salmonel


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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 digest

DNA 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 Arbers hypothesiswas correctthe 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 on

determining 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, polynucleotide

kinase, 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 first

base 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 Smiths 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 the

nucleotide sequence recognized by endonuclease R. T7 bacterio-

phage DNA was digested with endonulcease R. After removal of

the 5 phosphate, and addition of a 32P label, the 5 end-labeled

DNA was digested with a variety of nucleases. 32P-labeled

mononucleotides, dinucleotides, and trinucleotides were isolated

and analyzed to determine the recognition site sequence.

[Adapted from T. J. Kelly and H. O. Smith, 1970, J. Mol.Biol.

51:393.]


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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.

Discussion

Although 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 thmethylase that allows DNA from H. influenzae to escapcleavage. By using these sequence-specific restrictioenzymes, researchers could now cleave DNA at specifsites. The impact of restriction enzymes on biologicresearch over cannot be overstated. Early on, theenzymes were used for mapping plasmid and phage DNANow they are routinely used for probing the structure both specific genes and of DNA from individuals. In addtion, they are primary reagents in the construction of genexpression vectors, allowing DNA from different sourcto be cleaved at specific sequences, then joined with simlarly cleaved DNA. The results are seen everyday in labratories employing recombinant DNA technologies. 1978, Hamiliton Smith was awarded the Nobel Prize foPhysiology and Medicine in recognition of his powerfdiscovery.


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Background

A 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 animals 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. RalphBrinster 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 foreign gene incorporated into the mouse genome.

The Experiment

Brinsters challenge was to design the experiment in suca way that it could be easily and unequivocally demostrated that the mouse was making the foreign protein. Taccomplish this, Brinster chose to express an easily assayeenzyme rather than a protein of greater biological inteest in his first transgenic mouse. He chose the enzymthymidine kinase from the herpes simplex virus (HSV), thchoice of which offered several advantages. First, the gencame from a human virus; thus its sequence sufficientdiffered from the endogenous mouse gene allowing its i

tegration into the mouse genome to be readily demostrated. Second, the activity of thymidine kinase can beasily assayed by following the conversion of radioactivelabeled thymidine to thymidine monophosphate. Finallan inhibitor of the HSV thymidine kinase activity that donot inhibit the endogenous mouse enzyme was availablallowing the researchers to specifically monitor the actiity of the foreign protein.

Genes are expressed from DNA sequences upstream the protein-coding region called promoters. Promotecontrol where and when a gene is expressed. To expre


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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, then

cleaved it with a restriction endonuclease. They proceededto separate the DNA by agarose gel electrophoresiswhich separates DNA fragments on the basis of sizeand 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.

Discussion

Progress 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, Cell27:223231.]


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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 four

polypeptide 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 ONESOMATIC

REARRANGEMENT OF

IMMUNOGLOBULIN 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 heavchains exhibited a similar pattern. These findings suggestethat the genome contains a small number of C genes ana much larger group of V genes.

In 1965, W. Dryer and J. Bennett proposed that tw

separate genes, one V gene and one C gene, encode eacheavy chain and each light chain. Although this proposseemed logical, it violated the well-documented principthat each gene encodes a single polypeptide. To avoid thobjection, Dryer and Bennett suggested that a V and gene somehow were rearranged in the genome to formsingle gene, which then was transcribed and translateinto a single polypeptide, either a heavy or light Ig chaiIndirect support for this model came from DNhybridization studies showing that only a small number genes encoded Ig constant regions. However, until mo


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

The Experiment

Tonegawa 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 of

the 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 digestionand RNA-DNA hybridization to detect the DNA frag-ments.

150

200

250

cpm

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100

5 10 15

MIGRATION (cm)Top ofgel

Bottomof gel

Embryo DNA

Whole gene

3' end of gene

Whole gene

3' end of gene

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200

250

cpm

50

0

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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 during

development 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. Natl. 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 type

of 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


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probes in handone specific for the combined V Cgene and one specific for C aloneTonegawa 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 ofB cells, separate genes encoding the V and C regions arerearranged into a single DNA sequence encoding theentire light chain (Figure 2).

Discussion

The 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 variety of other alterations that allow the immune systeto create the diverse repertoire of antibodies necessary treact to any invading organism. Our current understaning of these mechanisms rests on the foundation oTonegawas fundamental discovery. For this work, hreceived the Nobel Prize for Physiology and Medicine 1987.

5'

VK CK

5' 3'

VK CK

Embryonic DNA

B-Cell DNA

Sch

Classic Experiments

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