classic experiments in molecular biology

C L A S S I C E X P E R I M E N T 3 . 1

BRINGING AN ENZYME BACK TO LIFEC. B. Anfinsen and E. Haber, 1961, Journal of Biological Chemistry 236:1362

By the 1950s, scientists realized thatDNA held the code that allowed pro-teins to be synthesized. Nevertheless,how a chain of amino acids folds intoa fully functional protein, with theproper three-dimensional structure, re-mained a mystery. A mechanism mustexist to assure the proper folding of theprotein. But where did that informa-tion come from? In 1957, ChristianAnfinsen published the first evidencethat the information for proper foldingwas held within the protein itself.

Background Proteins are made from combinationsof 20 amino acids that then fold intocomplex structures. The unfoldedamino acid chain is called the primarystructure. To have biological activity,the protein must fold into proper sec-ondary and tertiary structures. Thesestructures are held together by interac-tions between the side chains andbackbone atoms of the amino acids,including hydrogen bonds, hydropho-bic interactions, and, at times, covalentbonds. How these higher structuresform had long been a mystery. Doesthe protein fold correctly as it is syn-thesized or does it require the actionof other proteins to correctly fold it?Can it correctly fold on its own spon-taneously?

In the 1950s, Anfinsen was a bio-chemist interested in the proper fold-ing of proteins. Specifically, he wasinvestigating the formation of disulfidebridges, which are covalent bonds be-tween cysteine side chains that serveas one of the major anchors holdingtogether the structure of secreted pro-teins. He believed that the protein itselfcontained all the information neces-sary for proper protein folding. Heproposed the thermodynamic hypoth-esis, which stated that the biologicallyactive structure of a protein was also

the most thermodynamically stable un-der in vivo conditions. In other words,if the intracellular conditions could bemimicked in a test tube (in vitro), thena protein would naturally fold into itsactive conformation. He began his workon a secreted enzyme, bovine pancreaticribonuclease, and studied its ability toproperly fold outside of the cell.

The Experiment Proteins perform a wide variety of func-tions in the cell. Regardless of its func-tion, a protein must be properly foldedto carry out its biological role. For pro-tein folding studies it is best to study anenzyme whose biological activity can beeasily monitored by performing a test,or assay of its activity in vitro. Anfin-sen chose a small, secreted protein, theenzyme ribonuclease, in which he couldmonitor proper folding by assaying itsability to catalyze the cleavage of RNA.

Ribonuclease, a secreted protein, isactive under oxidizing conditions invitro. The tertiary structure of active ri-bonuclease is held together by fourdisulfide bonds or bridges. Adding a re-ducing agent reduces the disulfide bondbetween two cysteine side chains to twofree sulfhydryl groups, and can disruptthis covalent interaction. Completedenaturation of ribonuclease requirestreatment with a reducing agent. An-finsen monitored the reduction ofribonuclease by measuring the numberof free sulfhydryl groups present in theprotein. In the oxidized state, there areno free sulfhydryl groups in ribonucle-ase because each cysteine residue isinvolved in a disulfide bond. In thecompletely reduced state, on the otherhand, ribonuclease contains eight freesulfhydryl groups. Anfinsen exploitedthis difference to assess the extent of re-duction by using a spectrophotometricassay to titrate, or count, the number offree sulfhydryl groups.

To study protein folding outsidethe cell, one must first denature theprotein. Proteins are easily denaturedby heat, mechanical disruption such asshaking, and chemical treatment. Pro-teins with disulfide bridges require anadditional measure of treatment with areducing agent to break apart thesecovalent bonds. To denature ribonu-clease, Anfinsen first reduced the disul-fide bridges with thioglycolic acid. Hethen denatured the protein by using ahigh concentration of urea and incubat-ing the solution at room temperature.He demonstrated that this treatmentrendered the enzyme inactive by show-ing that ribonuclease was now unableto catalyze the cleavage of RNA. Usingthe spectrophotometric assay, he wenton to show that the inactive ribonucle-ase contained eight sulfhydryl groups,which corresponded to the four bro-ken disulfide bridges. With a com-pletely reduced, denatured protein inhand, Anfinsen then could ask: Can adenatured enzyme correctly fold invitro and become active again?

To find the answer, Anfinsen al-lowed a solution of reduced, denaturedribonuclease to oxidize. He removedthe urea from the denatured enzyme byprecipitation. Next, he resuspendedthe urea-free denatured ribonuclease ina buffered solution and incubated itfor two to three days. Exposure tomolecular oxygen in the atmosphereoxidized the cysteine residues. He thencompared the activity of this renaturedribonuclease to that of the native en-zyme. In initial experiments, 1219percent of the previously inactive pro-tein were able to catalyze the cleavageof RNA once again. Proteins aggregateat high concentrations, which makes itdifficult for them to fold properly. Bydecreasing the overall concentrationof ribonuclease in solution, Anfinsenshowed that up to 94 percent of theprotein could be refolded (see Table 1).

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The enzyme had folded back to its ac-tive conformation outside of the cell,demonstrating that the information forthe protein folding is contained in theprotein itself.

Discussion

Through careful experiments, Anfin-sen demonstrated that the informationrequired to properly fold a protein is

contained in its primary sequence. Hiscareful analysis of the chemistry of thisprocess answered a fundamental ques-tion in biology. He went on to demon-strate the cell-free refolding of otherenzymes, including proteins lackingdisulfide bridges. While it is possible toproperly fold a number of proteins out-side of the normal protein-processingmachinery in the cell, this process isgreatly accelerated in vivo by a numberof proteins. Anfinsen continued tostudy the protein-folding problem. Al-though the thermodynamic hypothe-sis does not hold true for all proteins,Anfinsens demonstration of the cell-free refolding of ribonuclease made amark on the field of biochemistry. In1972, he received the Nobel Prize forChemistry for his work.

TABLE 1 Cell-free Refolding of Ribonuclease

ACTIVITY AS A PERCENT OF EQUIVALENTCONCENTRATION OF PROTEIN (MG/ML) CONCENTRATION 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 Chemistry236:1362.]

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PROVING THAT DNA REPLICATION IS SEMICONSERVATIVEM. Meselson and W. F. Stahl, 1958, Proc. Natl Sci. USA 44:671

The discovery that the structure ofDNA is a double helix, containing twocomplementary strands of DNA, led toa number of hypotheses about howDNA might be replicated. Althoughthe possible replication mechanismswere relatively easy to deduce, provingwhich occurs in vivo was a more diffi-cult task. In 1958, Matthew Meselsonand Franklin Stahl used the newly de-veloped techniques of density-gradientcentrifugation to show that DNAreplication proceeds in a semiconserv-ative fashion.

BackgroundDuring the 1950s, scientists uncoveredmany biological facts we now take forgranted, beginning with the discoverythat genetic information is passed onthrough deoxyribonucleic acid (DNA),and continuing through the elucidationof DNAs three-dimensional structure.As the decade neared a close, biologistswere ready to study how DNA passedon genetic information from theparental to the progeny generation.

James Watson and Francis Crickhad hypothesized, on the basis of theirdouble-helical model of DNA, thatreplication occurs in a semiconserva-tive fashion. That is, the double helixunwinds, the original parental DNAstrands serve as templates to direct thesynthesis of the progeny strand, andeach of the replicated DNA duplexescontains one old (parental) strand, andone newly synthesized strand, calledthe daughter strand. Another hy-pothesis proposed at the time was con-servative replication, whereby afterreplication the parental strands formedone DNA duplex and the two daughterstrands formed the second duplex.

When these hypotheses were firstproposed, little experimental evidencewas available to support one over an-other. In 1957, however, Meselson and

Stahl, along with Jerome Vinograd, de-veloped density-gradient centrifuga-tion, a technique that can separatemacromolecules exhibiting very smalldifferences in density. The tools werenow available for a definitive test todetermine whether DNA replicationoccurs by a semiconservative or con-servative mechanism.

The ExperimentMeselson and Stahl reasoned that ifone could label the parental DNA insuch a way that it could be distin-guished from the daughter DNA, thereplication mechanisms could be distin-guished. If DNA replication is semicon-servative, then after a single round ofreplication, all DNA molecules shouldbe hybrids of parental and daughterDNA strands. If replication is conserva-tive, then after a single round of replica-tion, half of the DNA molecules shouldbe composed only of parental strandsand half of daughter strands.

To differentiate parental DNAfrom daughter DNA, Meselson andStahl used heavy nitrogen (15N).This isotope contains an extra neutronin its nucleus, giving it a higher atomicmass than the more abundant lightnitrogen (14N). Since nitrogen atomsmake up part of the purine and pyrim-idine bases in DNA, it was easy to la-bel E. coli DNA with 15N by growingthe bacteria in a medium containing15N ammonium salts as the sole nitro-gen source. After several generationsof growth, the bacteria contained only15N-labeled DNA. Now that theparental DNA was labeled, Meselsonand Stahl abruptly changed themedium to one containing 14N as thesole nitrogen source. From this pointon, all the DNA synthesized by thebacteria would incorporate 14N,rather than 15N, so that the daughterDNA strands would contain only

14N. As the bacteria continued togrow and replicate their DNA in the14N-containing medium, samples weretaken periodically and the bacterialDNA was analyzed with the newlydeveloped technique of equilibriumdensity-gradient centrifugation. In thistype of analysis, a DNA sample ismixed with a solution of cesium chlo-ride (CsCl2). During long periods ofhigh-speed centrifugation the CsCl2forms a gradient, and the DNA mi-grates to the position where the densityof the DNA is equal to that of theCsCl2. If the DNA sample containsmolecules of different densities theywill migrate to different positions inthe gradient. Because 15N has a greaterdensity than 14N, 15N-labeled DNAhas a greater density than 14N-labeledDNA. The higher-density (15N) DNAwill sediment to a different positionthan the lower-density (14N) DNA. Hy-brid DNA molecules, containing both15N and 14N, will sediment at an inter-mediate density, depending on the ratioof heavy nitrogen to light nitrogen.

The Figure illustrates the resultsobtained by Meselson and Stahl. Be-fore any DNA replication had oc-curred in the 14N-containing medium,all DNA sedimented as a single species,corresponding to 15N-labeled DNA.As DNA replication proceeded, theamount of (15N)-DNA decreased, anda second DNA species, consisting ofhybrid DNA molecules containing15N- and 14N-labeled strands, ap-peared. DNA collected after comple-tion of the first round of replicationwas found to sediment with the secondspecies. When the DNA produced dur-ing a second round of replication wasanalyzed, two distinct species were ob-served. One corresponded to hybridmolecules; the other corresponded to14N-labeled DNA. With each subse-quent round of replication the propor-tion of hybrid DNA decreased as the

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Experimental demonstration by Meselson and Stahl that DNAreplication is semiconservative. After several generations of growth ina medium containing heavy (15N) nitrogen, E. coli was transferredto a medium containing the normal light isotope (14N). Sampleswere removed from the cultures periodically and analyzed byequilibrium density-gradient centrifugation in CsCl to separate heavy-

heavy (H-H), light-light (L-L), and heavy-light (H-L) duplexes intodistinct bands. The actual banding patterns observed were consistentwith the semiconservative mechanism. [From H. Lodish et al., 1995,Molecular Cell Biology, 3rd ed. W. H. Freeman and Company. See M. Meselson

and W. F. Stahl, 1958, Proc. Natl. Acad. Sci. USA 44:671; photographs

courtesy of M. Meselson.]

Oldstrand

Parent strandssynthesizedin 15N

First doublingin 14N

Second doublingin 14N

One band:HH

Two bands:HH + LL

New strands

One band:HH

One band:HL (hybrid)

Two bands:HL + LL

Two bands:HH + LL

Light(14N)

Heavy(15N)

HHH H

LHHLLLHH

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


servative manner, they not only had todesign a clear, easily interpretable ex-periment, but also develop the technol-ogy to do it. The beauty of this classicexperiment is that each of the possiblemodels would produce distinctly differ-ent results, so that interpretation of theexperimental data was unambiguous.This study remains a shining exampleof defining a problem and employingthe proper methodology to solve it.

By demonstrating that DNA repli-cation occurs in a semiconservativefashion, Meselson and Stahl opened upthe field of DNA replication for in-depth research. With the correct modelin hand, researchers could now turn tounraveling the precise mechanism ofDNA replication. In addition, equilib-rium density-gradient centrifugationbecame a widely used tool for theanalysis of complex mixtures of DNA.

amount of 14N-labeled DNA in-creased. As the diagrams in the Figureshow, the sedimentation patterns ob-served by Meselson and Stahl are con-sistent only with a semiconservativemodel of replication.

DiscussionFor Meselson and Stahl to prove thatDNA replication proceeds in a semicon-



CRACKING THE GENETIC CODEM. W. Nirenberg and P. Leder, 1964, Science 145:1399

By the early 1960s molecular biologistshad adopted the so-called centraldogma, which states that DNA directssynthesis of RNA (transcription), whichthen directs assembly of proteins(translation). However, researchers stilldid not completely understand howthe code embodied in DNA andsubsequently in RNA directs proteinsynthesis. To elucidate this process,Marshall Nirenberg embarked upon aseries of studies that would lead to thesolution of the genetic code.

BackgroundProteins are made from combinationsof 20 different amino acids. The genesthat encode proteinsthat is, specifythe type and linear order of their com-ponent amino acidsare located inDNA, a polymer made up of only fourdifferent nucleotides. The DNA code istranscribed into RNA, which is alsocomposed of four nucleotides. Niren-bergs studies were premised on the hy-pothesis that the nucleotides in RNAform codewords, each of which cor-responds to one of the amino acidsfound in protein. During protein syn-thesis, these codewords are translatedinto a functional protein. Thus, to un-derstand how DNA directs proteinsynthesis, Nirenberg set out to under-stand the relationship between RNAcodewords and protein synthesis.

At the outset of his studies, muchwas already known about the processof protein synthesis, which occurs onribosomes. These large ribonuleopro-tein complexes can bind two differenttypes of RNA: messenger RNA (mRNA),which carries the exact protein-specifyingcode from DNA to ribosomes, andsmaller RNA molecules now known astransfer RNA (tRNA), which deliveramino acids to ribosomes. tRNAs existin two forms: those that are covalentlyattached to a single amino acid, known

as amino-acylated or charged tRNAs,and those that have no amino acidattached, called uncharged tRNAs.After binding of the mRNA and theamino-acylated tRNA to the ribosome,a peptide bond forms between theamino acids, beginning protein synthe-sis. The nascent protein chain is elon-gated by the subsequent binding ofadditional tRNAs and formation of apeptide bond between the incomingamino acid and the end of the growingchain. Although this general processwas understood, the question re-mained: How does the mRNA directprotein synthesis?

When attempting to address com-plex processes such as protein synthe-sis, scientists divide large questionsinto a series of smaller, more easily ad-dressed questions. Prior to Nirenbergsstudy, it had been shown that whenphenylalanine-charged tRNA was incu-bated with ribosomes and polyuridylicacid (polyU), peptides consisting ofonly phenylalanine were produced.This finding suggested that the mRNAcodeword, or codon, for phenylalanineis made up of the nucleosides contain-ing the base uracil. Similar studieswith polycytadylic acid (polyrC) andpolyadenylic acid (polyrA) showedthese nucleosides containing the basescytadine and adenine made up thecodons for proline and lysine, respec-tively. With this knowledge in hand,Nirenberg asked the question: What isthe minimum chain length required fortRNA binding to ribosomes? The sys-tem he developed to answer this ques-tion would give him the means to de-termine which amino-acylated tRNAwould bind which m-RNA codon,effectively cracking the genetic code.

The ExperimentThe first step in determining the mini-mum length of mRNA required for

tRNA recognition was to develop anassay that would detect this interac-tion. Since previous studies had shownthat ribosomes bind mRNA and tRNAsimultaneously, Nirenberg reasonedthat ribosomes could be used as a bridgebetween a known mRNA codon and aknown tRNA. When the three compo-nents of protein synthesis are incu-bated together in vitro, they shouldform a complex. After devising amethod to detect this complex,Nirenberg could then alter the size ofthe mRNA to determine the mini-mum chain length required for tRNArecognition.

Before he could begin his experi-ment, Nirenberg needed both a meansto separate the complex from unboundcomponents and a method to detecttRNA binding to the ribosome. To iso-late the complex he exploited the abil-ity of nylon filters to bind large RNAmolecules, such as ribosomes, but notthe smaller tRNA molecules. He used anylon filter to separate ribosomes (andanything bound to the ribosomes)from unbound tRNA. To detect thetRNA bound to the ribosomes,Nirenberg used tRNA charged withamino acids that contained a radioac-tive label, 14C. All other componentsof the reaction were not radioactive.Since only ribosome-bound tRNA isretained by the nylon membrane, allradioactivity found on the nylon mem-brane corresponds to tRNA bound toribosomes. Now a system was in placeto detect the recognition between amRNA molecule and the properamino-acylated tRNA.

To test his system, Nirenberg usedpolyU as the mRNA, and [14C]-phenylalanine-charged tRNA, whichbinds to ribosomes in the presence ofpolyU. Ribosomes were incubated withboth polyU and [14C]-phenylalaninetRNA for sufficient time to allow bothmolecules to bind to the ribosomes; the


reaction mixtures were then passedthrough a nylon membrane. When themembranes were analyzed using ascintillation counter, they containedradioactivity, demonstrating that inthis system polyU could recognizephenylalanine-charged tRNA. But wasthis recognition specific for the properamino-acylated tRNA? As a control,[14C]-lysine- and [14C]-proline-chargedtRNAs also were incubated withpolyU and ribosomes. After the reac-tion mixtures were passed through anylon filter, no radioactivity was de-tected on the filter. Therefore, the as-say measured only specific binding be-tween a mRNA and its correspondingamino-acylated tRNA.

Now the minimum chain lengthof RNA necessary for proper amino-acylated tRNA recognition could be de-termined. Short oligonucleotides weretested for their ability to bind ribosomesand recognize the appropriate tRNA.When UUU, a trinucleotide, was used,tRNA binding to ribosomes could be

detected. However, when the UU dinu-cleotides were used, no binding wasdetected. This result suggested that thecodon required for proper recognitionof tRNA is a trinucleotide. Nirenbergrepeated this experiment on two otherhomogeneous trinucleotides, CCC andAAA. When these trinucleotides wereindependently bound to ribosomes,CCC specifically recognized prolinecharged tRNA and AAA recognizedlysine-charged tRNAs. Since none ofthe three homogeneous trinucleotidesrecognized other charged tRNAs,Nirenberg concluded that trinu-cleotides could effectively direct theproper recognition of amino-acylatedtRNAs.

This study accomplished muchmore than determining the length ofthe codon required for proper tRNArecognition. Nirenberg realized thathis assay could be used to test all 64possible combinations of trinucleotides(see Figure). A method for cracking thecode was available!

Discussion

Combined with the technology to gen-erate trinucleotides of known se-quence, Nirenbergs assay provided away to assign each specific amino acidto one or more specific trinucleotides.Within a few years, the genetic codewas cracked, all 20 amino acids wereassigned at least one trinucleotide, and61 of the 64 trinucleotides were foundto correspond to an amino acid. Thefinal three trinucleotides, now knownas stop codons, signal terminationof protein synthesis.

With the genetic code cracked, bi-ologists could read the gene in thesame manner that the cell did. Simplyby knowing the DNA sequence of agene, scientists can now predict theamino acid sequence of the protein itencodes. For his innovative work,Nirenberg was awarded the NobelPrize in Physiology or Medicine in1968.

Assay developed by Marshall Nirenberg and his collaborators fordeciphering the genetic code. They prepared 20 E. coli extractscontaining 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 remainedunlabeled. Aminoacyl-tRNAs and trinucleotides passed through a nylonfilter without binding (left panel); ribosomes, however, bind to the filter(center panel). Each of the 64 possible trinucleotides was testedseparately for its ability to attract a specific tRNA by adding it with

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

ribosomes to different extract samples. Each sample was then filtered.If the added trinucleotide causes the radiolabeled aminoacyl-tRNA tobind to the ribosome, then radioactivity is detected on the filter;otherwise, the label passes through the filter (right panel). Bysynthesizing and testing all possible trinucleotides, the researchers wereable 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].



UNLEASHING THE POWER OF EXPONENTIAL GROWTHTHEPOLYMERASE CHAIN REACTIONR. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, N. Arnheim, 1985, Science 230(4732):13504

In the early 1980s the fruits of the mo-lecular biology revolution were begin-ning to be realized. Geneticists wereuncovering the genetic defects thatlead to many hereditary diseases, andthe newly burgeoning biotechnologyindustry was eager to provide physi-cians with simple diagnostic tests forsuch diseases. However, the bestmethod available for detecting abnor-mal genes, Southern hybridization,required sizable DNA samples andseveral days to perform. In this envi-ronment, one of the most powerfulmolecular biology techniques knownwas born: the polymerase chain reac-tion, or PCR.

BackgroundResearchers in the human genetics de-partment of a young biotechnologycompany were trying to develop apractical method for the prenatal diag-nosis of sickle-cell anemia. The molec-ular defect that causes most cases ofthis disease is a single nucleotidechange in the sixth codon of the geneencoding the protein -globin, one ofthe subunits of hemoglobin. KaryMullis, a molecular biologist at thecompany, had an idea for a molecularmethod that would amplifiy specificDNA sequences. The detection of asingle nucleotide change, as occurs insickle-cell anemia, was the perfect testfor his ideas.

Mulliss idea was an extension ofknown techniques for synthesizingspecific pieces of DNA in vitro usingchemically synthesized oligonucleotidesand purified DNA polymerase, theenzyme that catalyzes the synthesis ofDNA. First, a short oligonucleotidewhose sequence was complementaryto a portion of the target DNA wassynthesized. Next, a fragment of DNA

containing the target sequence was iso-lated using restriction endonucleases,enzymes that catalyzed the cleavage ofDNA at specific sequences. The iso-lated DNA fragment was then heatedto denature the double-stranded helixinto two single-stranded DNA mole-cules. At this point, the oligonucleotidewas added to the DNA and allowed toanneal to the complementary region,thereby creating a primer-templatecomplex, one of the substrates forDNA polymerase. The other sub-strates, the four deoxynucleotidetriphosphates (dNTPs), were thenadded, so that DNA synthesis couldoccur. Although this method wasuseful for producing radioactivelylabeled pieces of DNA, it could notamplify a DNA sequence, only repli-cate it.

The ExperimentMullis designed a method that wouldactually amplify the amount of targetDNA, a prerequisite for detecting asmall DNA sequence within a largecomplex sample of genomic DNA. Forinstance, the human genome conations3 109 nucleotides of coding se-quence. Molecular diagnosis of sickle-cell anemia requires the detection ofone altered nucleotide in one geneamong the rest of the genome. To ac-complish this, the region of thegenome containing the alteration mustbe amplified.

Basing his work on the sequence ofthe -globulin gene, which wasknown, Mullis designed primers thatwould anneal at sequences both up-stream and downstream from the dis-ease causing mutation. One primerwas complementary to the codingstrand, known as the () strand, thesecond was complementary to the non-

coding, or (), strand. When theprimers were added to a sample of de-natured genomic DNA along withDNA polymerase and the four dNTPs,DNA synthesis occurred across the re-gion of the mutation from both of theoriginal strands, producing two newdouble-stranded DNA molecules.Thus the DNA between the primersites was doubled, not simply repli-cated as in the older method. Mullis re-alized that each cycle of DNA-primerannealing and DNA synthesis wouldyield twice as much target DNA as theprevious cycle (see Figure). A chain re-action would ensue and the amount ofDNA in the sample would grow expo-nentially. He called his technique thepolymerase chain reaction (PCR) to re-flect the mechanism by which amplifi-cation was occurring.

The first published test of the PCRmade use of upstream and down-stream oligonucleotide primers thatflanked a 110-bp region of the -globin gene; the target region includedthe mutation found in sickle-cell ane-mia. These primers were mixed withsamples of amniotic fluid that hadbeen previously typed for the presenceor absence of the mutation. After thesamples were put through 20 cycles ofheat denaturation, cooling to allowannealing, and DNA synthesis or primerelongation, the amount of -globintarget DNA in the samples was foundto be enriched more than one milliontimes (220) compared with the initialsamples. The exponential expansion ofthe DNA was easily demonstrated bycomparing the same sample after 15and 20 cycles. It was clear that theadditional five cycles greatly in-creased the amount of DNA producedin the reaction. Next, Mullis testedthe ability of the PCR to detect smallquantities of DNA. He found that after


sequence could be amplified, not justreplicated, if synthesis were carriedout from both the coding and non-coding strands. Second, that a targetDNA sequence would grow likedividing bacteria in a culture if theamplification cycle was repeated severaltimes in succession. By employingthis relatively simply methodology,Mullis developed one of the mostpowerful techniques in molecularbiology.

The advantages of PCR were ob-vious from the first report. Almost

instantly, it became a standard tech-nique used in all fields of biology andmedicine, as well as the forensic sci-ences. Today, the technique is knownnot only to biologists, but also topeople in all walks of life. In 1993,just eight years after his first reporton the PCR, Kary Mullis wasawarded the Nobel Prize in Chem-istry for developing this revolution-ary technique.

20 cycles, the -globulin gene could bedetected starting with a genomic DNAsample as small as 20 ng, which was 50times smaller than the samples in theoriginal tests. This finding implied thatthe PCR could be used in a variety ofsituations where only a small amount ofDNA was available, contributing to thewidespread use of the technique today.

DiscussionDevelopment of the PCR relied ontwo key insights. First, that a DNA

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

53

35

33

Add DNA polymerase [ ]dNTPs

Heat denatureCool to allow primer annealingRepeat reaction

Repeat reaction for 20 cycles

DNA encoding the -globin gene

Upstream primer

Downstream primer

*

*

*

**

*

*

*

*

*

Nucleotide mutatedin sickle-cell anemia

*

DenatureAdd primers in excess



DEMONSTATING SEQUENCE-SPECIFIC CLEAVAGE BY A RESTRICTION ENZYMET. J. Kelly and H. O. Smith, 1970, J. Mol. Biol. 51:393

Bacteria exhibit a phenomenon, knownas host restriction, whereby they canboth recognize and cleave foreign DNA,preventing it from interfering with thebacterial life cycle. By purifying andcharacterizing one of the enzymes in-volved in host restriction, HamiltonSmith gave molecular biology one of itsmost important tools, an enzyme thatcleaves DNA at a specific sequence.

BackgroundAt the time of Hamilton Smiths work,host restriction was a well-characterizedyet highly intriguing phenomenon. Itwas well known that DNA from onespecies of bacteria could not be used totransform a second species of bacteria.When researchers simply mixed DNAfrom one bacterium with a lysate froma second bacterial species, the DNAwas cleaved. The bacteria had evolveda system to recognize and cleave for-eign DNA. In 1965, Werner Arber hy-pothesized that bacteria must producean enzyme capable of recognizing andcleaving foreign DNA at specific se-quences. How did a bacterium deter-mine which DNA was foreign, andwhich was its own? It seemed unlikelythat a bacterium could exclude specificsequences in its genome, from the ac-tion of this nuclease. More likely, abacterium somehow modified its ownDNA at these sequences, so it could bespared from cleavage. The existence ofa second enzyme was thus hypothe-sized, one that could modify the DNAby methylation at the site where cleav-age occurred, thereby preventing cleav-age by the sequence-specific nuclease.

With these hypotheses in hand, thehunt for the enzymes could begin. In1968, Matthew Meselson reported thepurification from E. coli of one of theseenzymes, now called restriction enzymes

or restriction endonucleases. Althoughthe E. coli enzyme catalyzed the cleav-age of non-E.coli DNA, Meselson couldnot demonstrate that this cleavage wassequence specific. In fact, proving thatthese bacterial enzymes cleave DNA at aspecific sequence would be a tricky mat-ter, as this research was conductedbefore the advent of the relatively simpleDNA-sequencing techniques now avail-able. Following on Meselsons work,Smith set out to purify a second restric-tion enzyme, this time from H. influen-zae, and to demonstrate that it doesindeed cleave DNA in a sequence-specific manner.

The ExperimentThe first step in the successful purifica-tion of a new enzyme is devising an as-say that measures the known activityof the enzyme as it is being purified.The activity of a restriction enzymecatalyzes the cleavage of foreign DNA,so this was the logical activity to mon-itor. To do so, Smith took advantage ofthe fact that genomic DNA from bac-teria is quite viscous; however, as nu-cleases begin to degrade the bacterialDNA, the overall viscosity decreases.Therefore, Smith could monitor thepurification of his restriction enzymeby measuring the decrease in viscosityof a foreign DNA after treatment witha sample of the protein after each stepin the purification scheme. Smithmixed cell extracts of H. influenzaewith intact DNA from either H. influen-zae or the Salmonella bacteriophageP22. Using a device called a viscome-ter, he measured how the DNA fromP22 became less viscous over time,while the H. influenzae DNA dis-played no change in viscosity. Thiswould be the assay he would usethroughout the purification scheme.

Smith used a variety of establishedmethods to separate bacterial lysatesinto smaller pools of proteins. Eachmethod separated the lysate on the basisof a different physical property of theproteins (and other biomolecules) thatmake up the lysate. This allowed thelysate to be divided into subsamplesknown as fractions. After each step inthe purification, every fraction was sep-arately assayed for the ability to cleaveP22 DNA. Fractions that contained theenzyme activity were subjected to yetanother purification method, and theprocess was continued until a pure en-zyme was obtained. Smith called the pu-rified restriction enzyme endonuclease R.

Next Smith determined some of thebasic characteristics of endonuclease R.He used endonuclease R to digest DNAfrom the bacteriophage T7, then esti-mated the number of sites where theDNA was cleaved. He discovered thatendonuclease R did not completely de-grade T7 DNA, but rather cleaved it atapproximately 40 sites. Since T7 DNAcontains approximately 40,000 bases,cleavage occurred at only 0.1 percentof the possible sites. This observationsuggested to Smith that Arbers hypoth-esis was correctthe enzyme wascleaving the DNA at specific sequences.In order to prove that this was the case,Smith had to determine the sequence atwhich the enzyme cleaved the DNA,which he called the recognition site.

With the purified enzyme and evi-dence of sequence-specific DNA cleav-age, Smith focused his attention ondetermining the sequence of the recog-nition site. At this time, the 1960s, theonly known method of DNA sequencingwas to sequentially remove nucleotidesfrom the 5 end of DNA and determinetheir identity by thin layer chromatog-raphy (TLC). Smith devised a schemeto sequence the recognition site by

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with endonuclease R, the 5 terminaldinucleotides are the first two bases inthe recognition site. Smith first sepa-rated the dinucleotides from the singlenucleotides. When he analyzed the din-ucleotides by TLC, he found only twospecies of dinucleotides that carried the32P label. The identity of the 32P-labeleddinucleotides was determined by com-paring their migration to that of dinu-cleotides of known sequence. One ofthe species displayed the same migra-tion as the dinucleotide GA; the othermigrated with the dinucleotide AA.Smith concluded that the second base inthe recognition sequence was adenine.

Analysis of the rest of the recogni-tion site would not be so easy, butSmiths persistence paid off. Using asimilar but slightly more complicatedmethod, he identified the third base inthe recognition site as cytosine. He fur-ther showed this to be the end of therecognition sequence by showing thatthe fourth nucleotide could contain anybase. Now he knew digestion of double-stranded DNA with endonuclease Rcreates several smaller fragments withidentical 5 ends, which contain the se-quence purine-adenine-cytosine. Sincethe DNA strands are complementary,the only possible way this could occur isif the enzyme recognized a six-basesequence that appeared the same oneither strand, known as a pallindromic

sequence. Therefore, Smith concludedthat endonuclease R recognized andcleaved DNA specifically at the se-quence GTPyPuAC.

DiscussionAlthough the first restriction enzymehad been purified two years beforeSmith reported his work on endonucle-ase R, he was the first to demonstratesequence-specific cleavage. He then wenton to purify and characterize the methy-lase that allows DNA from H. influen-zae to escape cleavage. By using thesesequence-specific restriction enzymes,researchers could now cleave DNA atspecific sites. The impact of restrictionenzymes on biological research cannotbe overstated. Early on, these enzymeswere used for mapping plasmid andphage DNA. Now they are routinelyused for probing the structure of bothspecific genes and of DNA fromindividuals. In addition, they are pri-mary reagents in the construction ofgene expression vectors, allowing DNAfrom different sources to be cleaved atspecific sequences, then joined with sim-ilarly cleaved DNA. The results are seeneveryday in laboratories employing re-combinant DNA technologies. In 1978,Hamiliton Smith was awarded the No-bel Prize in Physiology or Medicine inrecognition of his powerful discovery.

using known enzymes to cleave the endsof a DNA strand into small pieces thatcould be analyzed by TLC (see Figure).

Smith began by labeling the 5 endof endonuclease R-digested DNA witha radioactive marker, 32P. This was ac-complished by first treating the DNAwith alkaline phosphatase, an enzymethat catalyzes the removal of 5 phos-phate groups from polynucleotides.Next, polynucleotide kinase, whichcatalyzes addition of phosphate to the5 end of polynucleotides, was used totransfer 32P from labeled ATP to theterminal nucleotide. Now the terminalnucleotide could be easily distinguishedfrom the rest of the nucleotides, byvirtue of its specific radioactive label.The DNA was then digested to singlenucleotides with a nuclease called pan-creatic DNase. The only 32P-labelednucleotides observed contained ade-nine (A) and guanine (G). Since no 32P-labeled nucleotide containing cytosine(C) or thymine (T) was detected, Smithdeduced that the first base in the recog-nition sequence must be a purine.

To determine the second base in therecognition site, Smith used a nucleasethat could not cleave 5 terminal dinu-cleotides. In other words, the entireDNA sample was digested into singlenucleotides except the final two, whichremained in dinucleotide form. Sincethe DNA previously had been cleaved

Schematic representation of the method used to determine thenucleotide sequence recognized by endonuclease R. T7 bacteriophageDNA was digested with endonulcease R. After removal of the 5phosphate, and addition of a 32P label, the 5-end-labeled DNA wasdigested with a variety of nucleases. 32P-labeled mononucleotides,dinucleotides, and trinucleotides were isolated and analyzed todetermine the recognition site sequence. [Adapted from T. J. Kelly and H. O. Smith, 1970, J. Mol.Biol. 51:393.]

53

53

35

35

Endonuclease R

Alkaline phosphatase

Polynucleotide kinase[32P] ATP

Digestion with variousnucleases

PP

P**P

53

35

53

35

P* *P

*P

*P

Recognition site

Mononucleotides

Dinucleotides

Trinucleotidesn = 3

P*n = 2

P*n = 1

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EXPRESSING FOREIGN GENES IN MICER. L. Brinster et al., 1981, Cell 27:223231

In the span of three years, 19801982,the notion of expressing foreign pro-teins in mice went from an idea to a re-ality. During this time, several labora-tories worked furiously to introducenew genes and express exogenous pro-teins, first in mouse embryonic stemcells and then in full-grown mice. RalphBrinster and Richard Palmiter wereamong the pioneers in this field when,in 1981, they first demonstrated therobust expression of a viral gene in atransgenic mouse.

BackgroundA powerful approach to the study ofgenes and the proteins they encode isthe controlled expression in both cellsand whole organisms. Before the ad-vent of recombinant DNA techniques,biologists accomplished this by inject-ing foreign mRNA into oocytes fromfrogs and studying the biological activ-ity of the protein encoded by the for-eign mRNA. In the 1970s and 1980s,the molecular biology revolution al-lowed genes to be fused to specific pro-moters, which would allow them to beexpressed in cell line. Whereas biologistsbecame able to study the gene functionin cultured cells, they still wanted tostudy genes in a living organism. Thisrequires the expression of a specific for-eign gene in embryonic cells, leading tointroduction of the foreign gene into theanimals genome, and examination of itsfunction in the organism.

In the early 1970s, Brinster demon-strated that foreign genes could be ex-pressed in mice by injecting cancercells into an early embryonic formknown as a blastocyst. This approach,however, made it difficult to express aspecific gene in the desired cell types.This would require introducing thegene into the mouse genome. In 1980,biologists demonstrated that this waspossible by injecting a plasmid con-

taining viral DNA into fertilizedmouse oocytes, then detecting the viralsequences in the newborn mice. Thisset the stage to determine whether afunctional protein could be expressedfrom a foreign gene incorporated intothe mouse genome.

The ExperimentBrinsters challenge was to design theexperiment in such a way that it couldbe easily and unequivocally demon-strated that the mouse was making theforeign protein. To accomplish this,Brinster chose to express an easily as-sayed enzyme rather than a protein ofgreater biological interest in his firsttransgenic mouse. He chose the en-zyme thymidine kinase from the herpessimplex virus (HSV), the choice ofwhich offered several advantages.First, the gene came from a humanvirus; thus its sequence sufficiently dif-fered from the endogenous mouse geneto allow its integration into the mousegenome to be readily demonstrated.Second, the activity of thymidine ki-nase can be easily assayed by followingthe conversion of radioactively labeledthymidine to thymidine monophos-phate. Finally, an inhibitor of the HSVthymidine kinase activity that does notinhibit the endogenous mouse enzymewas available, allowing the researchersto specifically monitor the activity ofthe foreign protein.

Genes are expressed from DNA se-quences upstream of protein-codingregions called promoters. Promoterscontrol where and when a gene is ex-pressed. To express a viral gene in amouse requires that the biologist re-move the gene from the control of theviral promoter and fuse it to a pro-moter that is active in mouse cells.Brinster collaborated with Palmiter,who had been studying the promoterof the mouse metallothionein-1 (MT-1)

gene. Palmiter fused the MT-1 pro-moter to the HSV thymidine kinasegene. They then could ask whether aviral protein could be expressed in amouse.

To generate the transgenic mouse,Brinster and Palmiter injected the plas-mid containing HSV thymidine kinasefused to the MT-1 promoter into thepro-nuclei of fertilized mouse eggs,which they then implanted back intofemale mice. The scientists matedprogeny mice with normal females,and analyzed the resulting progeny forthe presence of the HSV thymidine ki-nase DNA as well as thymidine kinaseactivity.

Using Southern blot analysis, theydetected the presence of the MT-1promoterthymidine kinase genefusion, known as the transgene. Theyisolated genomic DNA, then cleaved itwith a restriction endonuclease. Theyproceeded to separate the DNA byagarose gel electrophoresiswhichseparates DNA fragments on the basisof sizeand transferred it to a nitro-cellulose membrane. The two scientiststhen hybridized a radioactively labeledprobe, specific for the transgene, to themembrane for analysis. This analysisrevealed that the transgene had beensuccessfully integrated into the genomesof four progeny mice.

Next, to determine whether thetransgene expressed a functional pro-tein, Brinster and Palmiter analyzedhomogenates from the liver, a tissuewhere the mouse MT-1 gene is highlyexpressed, for viral thymidine kinaseactivity. Liver homogenates from onemouse contained approximately 200times more thymidine kinase activitythan the liver homogenates of its litter-mates. This mouse was one of the fourthat had the transgene integrated intoits genome. To demonstrate that thisincrease in activity was a result of viralthymidine kinase expression they treated


the presence of viral thymidine kinaseactivity and demonstrated that a foreignprotein could be expressed in a mouse.

DiscussionProgress in embryology and molecularbiology had left the field ripe for re-searchers to experiment with advanc-

ing the expression of foreign proteinsin animals. The careful choice of theeasily assayed HSV thymidine kinasegene put under the control of themetallothionein promoter allowedBrinster and Palmiter to demonstratethe feasibility of this technique.

The ability to generate transgenicmice has been invaluable to the studyof gene function in vivo. Before thistechnology was available, researchershad to find naturally occurring muta-tions in order to analyze gene functionin mice. Now a specific gene could beexpressed in mice. Soon genes werefused to promoters that allowed ex-pression in specific tissues. Scientistshave generated transgenic mice to ana-lyze the function of a great number ofgenes, allowing them to determine theroles of the genes in a variety of dis-eases and biological processes.

liver homogenates with an inhibitor thatspecifically blocks the HSV thymidinekinase activity. Thymidine kinase activityin liver homogenates from the transgenicmouse was markedly reduced by thisinhibitor, whereas the activity in ho-mogenates from its nontransgenic lit-termates was unchanged (see Table).Thus Brinster and Palmiter confirmed

Expression of Viral Thymidine Kinase in Transgenic Mice

THYMIDINE KINASE ACTIVITY

MOUSE TRANSGENE DNA INHIBITOR INHIBITOR

23-1 14500 14700

23-2 497,000 187,000

[Adapted from R. L. Brinster et al., 1981, Cell 27:223231.]



CATALYSIS WITHOUT PROTEINSTHE DISCOVERY OF SELF-SPLICING RNAK. Kruger et al., 1982, Cell 31:147

For biological systems to function,countless reactions must be catalyzed.These duties are carried out by en-zymes, biological macromolecules thatreadily enhance reaction rates yet re-main unconsumed by the reaction. Formany years only proteins were believedto possess sufficient diversity of func-tional groups to catalyze the myriadreactions necessary to sustain life. Then,in 1981, Thomas Cech reported that, inat least one case, RNA could do the job.

BackgroundIn eukaryotes and many viruses, genescontain sequences that are initiallytranscribed, then subsequently re-moved from RNA as they are not partof the actual coding sequence. Thesesequences are known as interveningsequences (IVS) or introns. IVS are re-moved from precursor RNA by a bio-logical process known as splicing.While investigating the splicing of pre-cursor ribosomal RNA (pre-rRNA)genes, transcribed from rRNA genes,Cech made his critical discovery thatRNA exhibited catalytic activity.

Cech wanted to understand themolecular components of RNA splic-ing. Rather than examing complex eu-karyotic genes, he chose a simplemodel system, rRNA genes from theciliated protozoan Tetrahymena ther-mophilia. By isolating Tetrahymenanuclei, Cech and his coworkers devel-oped a system in which pre-rRNA genesplicing could be studied in vitro. Thepurified nuclei could perform bothtranscription of rRNA genes and pro-cessing of the large pre-rRNA thatinitially is formed. Using this system,Cech found that during synthesis of26s rRNA in Tetrahymena, a 0.4-kbIVS is removed. The next step was toperform pre-rRNA splicing with

nuclear extracts, with an eye towardpurifying the enzymes that catalyzedthe splicing reaction. Although Cechsucceeded in this goal, he could havenever guessed how the catalysis wastaking place.

The ExperimentCechs plan was to use the in vitrosplicing system to purify the RNA-splicing enzymes, a common experi-mental approach for dissecting com-plex molecular processes. First, thereaction is characterized in a cell-freesystem, in this case purified nuclei.Then a means to purify the reactionsubstrate is developed. In the case ofthe Tetrahymena rRNA splicing thiswas relatively easy, because the full-length rRNA (pre-rRNA) transcriptswere abundant in Tetrahymena nucleiand readily purified. Finally, cellularextracts are added back to reconstitutethe activity being studied. Since theRNA splicing activity was known totake place in the nucleus, Cech used nu-clear extracts. In fact, he could readilysee splicing when nuclear extracts wereadded to rRNA transcripts in a splicingcocktail composed of Mg2 andguanosine triphosphate (GTP). Unex-pectedly, splicing also occurred whenrRNA transcripts were incubated in thesplicing cocktail in the absence of a nu-clear extract. This activity was repro-ducible, leaving open two possibilities:Either the purified pre-rRNA remainedassociated with an enzyme (i.e., a pro-tein contaminant) or the pre-rRNAwas catalyzing its own splicing.

The first step in determining whichpossibility was correct was to see if therRNA transcripts were truly devoid ofprotein. Because proteins are notori-ously fragile biomolecules, whoseactivity is easily destroyed by heat,

chemicals, and proteolytic enzymes,Cech subjected the rRNA transcriptsto numerous treatments known todegrade proteins: first, boiling to pro-mote heat denaturation; then, extrac-tion with organic solvents to promotechemical denaturation; finally, incuba-tion with a variety of proteases to pro-mote enzymatic degradation. Still, thepre-rRNA retained its splicing activity.These results strongly suggested thatTetrahymena pre-rRNA is indeed self-splicing. But a more definitive experi-ment was needed to convince other re-searchers that the transcripts wereuncontaminated by protein and pos-sessed inherent catalytic activity.

Fortunately, the Tetrahymena pre-rRNA could be produced in vitro usingpurified RNA polymerase from E. coli.Transcription of the TetrahymenarRNA gene with a polymerase from adifferent organism would eliminate therisk that the RNA remained associatedwith a Tetrahymena enzyme. In thissystem, the only enzyme ever associ-ated with RNA would be E. coliRNA polymerase, which was readilyremoved by extraction with organicsolvents. Using this system, Cech care-fully synthesized the Tetrahymena pre-rRNA, removed the polymerase, andpurified the transcripts. When he incu-bated this in vitro synthesized pre-rRNA in the splicing cocktail, analysisof the products showed that onceagain, the IVS was removed from theprecursor (see Figure). This experi-ment proved that the Tetrahymenapre-RNA was self-splicing, catalyzingthe removal of the IVS without the aidof any protein.

DiscussionCech called his self-splicing RNA aribozyme, implying that it was an


then turning over to splice others. Thisconvinced even the skeptics that RNAcan have true catalytic activity. Soonother self-splicing RNAs and other cat-alytic RNAs were identified. RNAcatalysis has become a field of studyunto itself, with research on the use ofcatalytic RNA in both laboratory andmedical settings. Furthermore, the

ability of RNA to catalyze biologicalreactions has evolutionary implica-tions. It is now conceivable that pri-mordial organisms contained onlyRNA and later evolved the morecomplex system of proteins. For hispioneering work on RNA catalysis,Cech was awarded the Nobel Prize inChemistry in 1989.

RNA enzyme. Although the demonstra-tion of self-splicing RNA was readilyaccepted by the scientific community,many were skeptical about the notionthat RNA was a true catalyst. In subse-quent studies, however, Cech was ableto engineer the Tetrahymena rRNAIVS such that it could be used as an en-zyme, splicing one RNA molecule,

Demonstration that Tetrahymena thermophilia pre-rRNA can self-splice. Radioactivelylabeled pre-rRNA was synthesized in vitro using E. coli RNA polymerase and thenincubated in neutral buffer or in the presence of Mg2 and GTP, necessary cofactorsfor the splicing reaction. Depicted here is an autoradiograph of the electrophoresedsamples revealing the spliced-out IVS in the sample containing splicing cofactors.[Adapted from Kruger et al., 1982, Cell 31:147.]

IVS

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



SEPARATING ORGANELLESBeaufay et al., 1964, Biochem J. 92:191

In the 1950s and 1960s scientists usedtwo techniques to study cell organelles:microscopy and fractionation. Christ-ian de Duve was at the forefront of cellfractionation. In the early 1950s heused centrifugation to distinguish anew organelle, the lysosome, frompreviously characterized fractions: thenucleus, the mitochondrial-rich frac-tion, and the microsomes. Soon there-after he used equilibrium-density cen-trifugation to uncover yet anotherorganelle.

Background Eukaryotic cells are highly organizedand composed of cell structuresknown as organelles that performspecific functions. Although mi-croscopy has allowed biologists to de-scribe the location and appearance ofvarious organelles, it is of limited usein uncovering an organelles function.To do this, cell biologists have reliedon a technique known as cell fraction-ation. Here, cells are broken open,and the cellular components are sepa-rated on the basis of size, mass, anddensity using a variety of centrifuga-tion techniques. Scientists could thenisolate and analyze cell componentsof different densities, called fractions.Using this method, biologists haddivided the cell into four fractions:nuclei, mitochondrial-rich fraction,microsomes, and cell sap.

De Duve was a biochemist inter-ested in the subcellular locations ofmetabolic enzymes. He had alreadycompleted a large body of work on thefractionation of liver cells, in which hehad determined the subcellular loca-tion of numerous enzymes. By locatingthese enzymes in specific cell fractions,he could begin to elucidate the func-tion of the organelle. He noted that his

work was guided by two hypotheses:the postulate of biochemical homo-geneity and the postulate of singlelocation. In short, these hypothesespropose that the entire composition ofa subcellular population will containthe same enzymes and that each en-zyme is located at a discrete site withinthe cell. Armed with these hypothesesand the powerful tool of centrifuga-tion, de Duve further subdivided themitochondrial-rich fraction. First, heidentified the light mitochondrial frac-tion, which is made up of hydrolyticenzymes that are now known to com-pose the lysosome. Then, in a series ofexperiments described here, he identi-fied another discrete subcellular frac-tion, which he called the peroxisome,within the mitochondrial-rich fraction.

The Experiment De Duve studied the distribution ofenzymes in rat liver cells. Highly ac-tive in energy metabolism, the livercontains a number of useful enzymesto study. To look for the presence ofvarious enzymes during the fractiona-tion, de Duve relied on known tests,called enzyme assays, for enzymeactivity. To retain maximum enzymeactivity, he had to take precautions,which included performing all frac-tionation steps at 0C because heatdenatures protein, compromising en-zyme activity.

De Duve used rate-zonal centrifu-gation to separate cellular componentsby successive centrifugation steps. Heremoved the rats liver and broke itapart by homogenization. The crudepreparation of homogenized cells wasthen subjected to relatively low-speedcentrifugation. This initial step sepa-rated the cell nucleus, which collects assediment at the bottom of the tube,

from the cytoplasmic extract, whichremains in the supernatant. Next, deDuve further subdivided the cytoplas-mic extract into heavy mitochondrialfraction, light mitochondrial fraction,and microsomal fraction. He accom-plished separating the cytoplasm byemploying successive centrifugationsteps of increasing force. At each stephe collected and stored the fractionsfor subsequent enzyme analysis. Oncethe fractionation was complete, deDuve performed enzyme assays to de-termine the subcellular distribution ofeach enzyme. He then graphically plot-ted the distribution of the enzymethroughout the cell. As had beenshown previously, the activity of cy-tochrome oxidase, an important en-zyme in the electron transfer system,was found primarily in the heavy mito-chondrial fractions. The microsomalfraction was shown to contain anotherpreviously characterized enzyme,glucose-6-phosphatase. The light mito-chondrial fraction, which is made upof the lysosome, showed the character-istic acid phosphatase activity. Unex-pectedly, de Duve observed a fourthpattern when he assayed uricase activ-ity. Rather than following the patternof the reference enzymes, uricase activ-ity was sharply concentrated withinthe light mitochondrial fraction. Thissharp concentration, in contrast to thebroad distribution, suggested to deDuve that the uricase might be se-cluded in another subcellular popula-tion separate from the lysosomalenzymes.

To test this theory, de Duve em-ployed a technique known as equilib-rium density-gradient centrifugation,which separates macromolecules onthe basis of density. Equilibriumdensity-gradient centrifugation can beperformed using a number of different


gradients, including sucrose and glyco-gen. In addition, the gradient can bemade up in either water or heavy wa-ter, which contains the hydrogen iso-tope deuterium in place of hydrogen.In his experiment de Duve separatedthe mitochondrial-rich fraction pre-pared by rate-zonal centrifugation ineach of these different gradients (Fig-ure 1). If uricase were part of a sepa-rate subcellular compartment, it wouldseparate from the lysosomal enzymesin each gradient tested. De Duve per-formed the fractionations in this seriesof gradients, then performed enzymeassays as before. In each case, he founduricase in a separate population thanthe lysosomal enzyme acid phos-phatase and the mitochondrial enzymecytochrome oxidase (Figure 2). By re-peatedly observing uricase activity in adistinct fraction from the activity ofthe lysosomal and mitochondrial en-zymes, de Duve concluded that uricasewas part of a separate organelle. Theexperiment also showed that two otherenzymes, catalase and Damino acid

FIGURE 2 Graphical representation of the enzyme analysis of products from asucrose gradient. The mitochondrial-rich fraction was separated as depicted in Figure 10.1,and then enzyme assays were performed. The relative concentration of active enzyme isplotted on the y axis; the height in the tube is plotted on the x axis. The peak activities ofcytochrome oxidase (top) and acid phosphatase (bottom) are observed near the top of thetube. The peak activity of uricase (middle) migrates to the bottom of the tube. [Adapted fromBeaufay et al., 1964, Biochem J. 92:191.]

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

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20 40 60 80

3

1

2

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20 40 60 80

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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 1 Schematic depiction ofthe separation of the lysosomes,mitochondria, and peroxisomes byequilibrium density centrifugation. Themitochondrial-rich fraction from rate-zonalcentrifugation was separated in a sucrosegradient, and the organelles were separatedon the basis of density. [From Lodish et al.,Molecular Cell Biology, 3d ed., W. H. Freeman and

Company, p. 166.]

oxidase, segregated into the same frac-tions as uricase. Because each of theseenzymes either produced or used hy-drogen peroxide, de Duve proposedthat this fraction represented anorganelle responsible for the peroxidemetabolism and dubbed it the peroxi-some.

Discussion De Duves work on cellular fractiona-tion provided an insight into the func-tion of cell structures as he sought tomap the location of known enzymes.Examining the inventory of enzymes ina given cell fraction gave him clues to

its function. His careful work resultedin the uncovering of two organelles:the lysosome and the peroxisome. Hiswork also provided important clues tothe organelles function. The lysosome,where de Duve found so many poten-tially destructive enzymes, is nowknown to be an important site fordegradation of biomolecules. The per-oxisome has been shown to be the siteof fatty acid and amino acid oxidation,reactions that produce a large amountof hydrogen peroxide. In 1974, deDuve received the Nobel Prize forPhysiology and Medicine in recogni-tion of his pioneering work.


C L A S S I C E X P E R I M E N T 1 1 . 1

STUMBLING UPON ACTIVE TRANSPORTJ. Skou, 1957, Biochem. Biophys. Acta 23:394

In the mid-1950s Jens Skou was ayoung physician researching the effectsof local anesthetics on isolated lipid bi-layers. He needed an easily assayedmembrane-associated enzyme to use asa marker in his studies. What he dis-covered was an enzyme critical to themaintenance of membrane potential,the Na/K ATPase, a molecularpump that catalyzes active transport.

BackgroundDuring the 1950s many researchersaround the world were actively investi-gating the physiology of the cell mem-brane, which plays a role in a numberof biological processes. It was wellknown that the concentration of manyions differs inside and outside the cell.For example, the cell maintains alower intracellular sodium (Na) con-centration and higher intracellularpotassium (K) concentration than isfound outside the cell. Somehow themembrane can regulate intracellularsalt concentrations. Additionally,movement of ions across cell mem-branes had been observed, suggestingthat some sort of transport is system ispresent. To maintain normal intracel-lular Na and K concentrations, thetransport system could not rely on pas-sive diffusion because both ions mustmove across the membrane againsttheir concentration gradients. Thisenergy-requiring process was termedactive transport.

At the time of Skous experiments,the mechanism of active transport wasstill unclear. Surprisingly, Skou had nointention of helping to clarify the field.He found the Na/K ATPase com-pletely by accident in his search for anabundant, easily measured enzyme ac-tivity associated with lipid membranes.A recent study had shown that mem-

branes derived from squid axons con-tained a membrane-associated enzymethat could hydrolyze ATP. Thinking thatthis would be an ideal enzyme for hispurposes, Skou set out to isolate such anATPase from a more readily availablesource, crab leg neurons. It was duringhis characterization of this enzyme thathe discovered the proteins function.

The ExperimentSince the original goal of his study wasto characterize the ATPase for use insubsequent studies, Skou wanted toknow under what experimental condi-tion its activity was both robust andreproducible. As often is the case withthe characterization of a new enzyme,this requires careful titration of thevarious components of the reaction.Before this can be done, one must besure the system is free from outsidesources of contamination.

In order to study the influence ofvarious cations, including three thatare critical for the reactionNa, K,and Mg2Skou had to make surethat no contaminating ions werebrought into the reaction from anothersource. Therefore all buffers used inthe purification of the enzyme wereprepared from salts that did not con-tain these cations. An additionalsource of contaminating cations wasthe ATP substrate, which containsthree phosphate groups, giving it anoverall negative charge. Because stocksolutions of ATP often included acation to balance the charge, Skouconverted the ATP used in his reac-tions to the acid form so that balancingcations would not affect the experi-ments. Once he had a well-controlledenvironment, he could characterize theenzyme activity. These precautionswere fundamental to his discovery.

Skou first showed that his enzymecould indeed catalyze the cleavage ofATP into ADP and inorganic phos-phate. He then moved on to look forthe optimal conditions for this activityby varying the pH of the reaction, andthe concentrations of salts and othercofactors, which bring cations into thereaction. He could easily determine apH optimum as well as an optimalconcentration of Mg2, but optimizingNa and K proved to be more diffi-cult. Regardless of the amount of K

added to the reaction, the enzyme wasinactive without Na. Similarly, with-out K, Skou observed only a low-level ATPase activity that did notincrease with increasing amounts ofNa.

These results suggested that the en-zyme required both Na and K foroptimal activity. To demonstrate thatthis was the case, Skou performed a se-ries of experiments in which he meas-ured the enzyme activity as he variedboth the Na and K concentrationsin the reaction (Figure 1). Althoughboth cations clearly were required forsignificant activity, something interest-ing occurred at high concentrations ofeach cation. At the optimal concentra-tion of Na and K, the ATPase activ-ity reached a peak. Once at that peak,further increasing the concentrationdid not affect the ATPase activity. Na

thus behaved like a classic enzyme sub-strate, with increasing input leading toincreased activity until a saturatingconcentration was achieved, at whichthe activity plateaued. K, on theother hand, behaved differently. Whenthe K concentration was increasedbeyond the optimum, ATPase activitydeclined. Thus while K was requiredfor optimal activity, at high concentra-tions it inhibited the enzyme. Skoureasoned that the enzyme must have


separate binding sites for Na andK. For optimal ATPase activity, bothmust be filled. However, at high con-centrations K could compete for theNa-binding site, leading to enzymeinhibition. He hypothesized that thisenzyme was involved in active trans-port, that is, the pumping of Na outof the cell, coupled to the import of K

into the cell. Later studies would provethat this enzyme was indeed the pumpthat catalyzed active transport. Thisfinding was so exciting that Skou de-voted his subsequent research to study-ing the enzyme, never using it as amarker, as he initially intended.

DiscussionSkous finding that a membrane AT-Pase used both Na and K as sub-strates was the first step in understand-ing active transport on a molecularlevel. How did Skou know to test bothNa and K? In his Nobel lecture in1997, he explained that in his first at-tempts at characterizing the ATPase,he took no precautions to avoid theuse of buffers and ATP stock solutionsthat contained Na and K. Ponderingthe puzzling and unreproducible re-sults that he obtained led to the real-ization that contaminating salts mightbe influencing the reaction. When he

repeated the experiments, this timeavoiding contamination by Na and Kat all stages, he obtained clear-cut, re-producible results.

The discovery of the Na/K

ATPase had an enormous impact onmembrane biology, leading to a betterunderstanding of the membrane poten-tial. The generation and disruption ofmembrane potential forms the basis ofmany biological processes, includingneurotransmission and the coupling ofchemical and electrical energy. For thisfundamental discovery, Skou wasawarded the Nobel Prize for Chem-istry in 1997.

FIGURE 1 Demonstration of the dependence of the Na/K

ATPase activity on the concentration of each ion. The graph onthe left shows that increasing K leads to an inhibition of the ATPaseactivity. The graph on the right shows that with increasing Na, the

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enzyme activity increases up to a peak and then levels out. This graphalso demonstrates the dependence of the activity on low levels ofK. [Adapted from J. Skou, 1957, Biochem. Biophys. Acta 23:394.]



FOLLOWING A PROTEIN OUT OF THE CELL J. Jamieson and G. Palade, 1966, Proc. Natl. Acad. Sci. USA 55(2):424431

The advent of electron microscopy al-lowed researchers to see the cell and itsstructures at an unprecedented level ofdetail. George Palade utilized this toolnot only to look at the fine details ofthe cell but also to analyze the processof secretion. By combining electronmicroscopy with pulse-chase experi-ments, Palade uncovered the path pro-teins follow to leave the cell.

Background In addition to synthesizing proteins tocarry out cellular functions, many cellsmust also produce and secrete addi-tional proteins that perform their du-ties outside the cell. Cell biologists,including Palade, wondered how se-creted proteins make their passagefrom the inside to the outside of thecell. Early experiments suggesting thatproteins destined for secretion are syn-thesized in a particular intracellular lo-cation and then follow a pathway tothe cell surface employed methods todisrupt cells synthesizing a particularsecreted protein and to separate theirvarious organelles by centrifugation.These cell-fractionation studies showedthat secreted proteins can be found inmembrane-bounded vesicles derivedfrom the endoplasmic reticulum (ER),where they are synthesized, and withzymogen granules, from which theyare eventually released from the cell.Unfortunately, results from these stud-ies were hard to interpret due to diffi-culties in obtaining clean separation ofall of the different organelles that con-tain secretory proteins. To further clar-ify the pathway, Palade turned to anewly developed technique, high-reso-lution autoradiography, that allowedhim to detect the position of radioac-tively labeled proteins in thin cell sec-tions that had been prepared for elec-

tron microscopy of intracellular or-ganelles. His work led to the seminalfinding that secreted proteins travelwithin vesicles from the ER to theGolgi complex and then to the plasmamembrane.

The Experiment Palade wanted to identify which cellstructures and organelles participate inprotein secretion. To study such acomplex process, he carefully chose anappropriate model system for his stud-ies, the pancreatic exocrine cell, whichis responsible for producing and se-creting large amounts of digestive en-zymes. Because these cells have theunusual property of expressing onlysecretory proteins, a general label fornewly synthesized protein, such asradioactively labeled leucine, will onlybe incorporated into protein moleculesthat are following the secretorypathway.

Palade first examined the proteinsecretion pathway in vivo by injectinglive guinea pigs with [3H]-leucine,which was incorporated into newlymade proteins, thereby radioactivelylabeling them. At time points from4 minutes to 15 hours, the animalswere sacrificed, and the pancreatic tis-sue was fixed. By subjecting the speci-mens to autoradiography and viewingthem in an electron microscope, Paladecould trace where the labeled proteinswere in cells at various times. As ex-pected, the radioactivity localized invesicles at the ER at time points imme-diately following the [3H]-leucine in-jection and at the plasma membrane atthe later time points. The surprisecame in the middle time points. Ratherthan traveling straight from the ER tothe plasma membrane, the radioac-tively labeled proteins appeared to

stop off at the Golgi complex in themiddle of their journey. In addition,there never was a time point where theradioactively labeled proteins were notconfined to vesicles.

The observation that the Golgicomplex was involved in protein secre-tion was both surprising and intrigu-ing. To thoroughly address the role ofthis organelle in protein secretion,Palade turned to in vitro pulse-chaseexperiments, which permitted moreprecise monitoring of the fate oflabeled proteins. In this labelingtechnique, cells are exposed to radiola-beled precursor, in this case [3H]-leucine, for a short period known asthe pulse. The radioactive precursor isthen replaced with its nonlabeled formfor a subsequent chase period. Proteinssynthesized during the pulse periodwill be labeled and detected by autora-diography, whereas those synthesizedduring the chase period, which arenonlabeled, will not be detected.Palade began by cutting guinea pigpancreas into thick slices, which werethen incubated for 3 minutes in mediacontaining [3H]-leucine. At the end ofthe pulse, he added excess unlabeledleucine. The tissue slices were then ei-ther fixed for autoradiography or usedfor cell fractionation. To ensure thathis results were an accurate reflectionof protein secretion in vivo, Palademeticulously characterized the system.Once convinced that his in vitro sys-tem accurately mimicked protein se-cretion in vivo, he proceeded to thecritical experiment. He pulse-labeledtissue slices with [3H]-leucine for 3minutes, then chased the label for 7,17, 37, 57, and 117 minutes with unla-beled leucine. Radioactivity, againconfined in vesicles, began at the ER,then traveled in vesicles to the Golgicomplex and remained in the vesicles


as they passed through the Golgi andonto the plasma membrane (see Fig-ure 1). As the vesicles traveled fartheralong the pathway, they became moredensely packed with radioactive pro-tein. From his remarkable series of au-toradiograms at different chase times,Palade concluded that secreted pro-teins travel in vesicles from the ER tothe Golgi and onto the plasma mem-brane and that throughout thisprocess, they remain in vesicles and donot mix with the rest of the cell.

Discussion Palades experiments gave biologiststhe first clear look at the stages of the

secretory pathway. His studies on pan-creatic exocrine cells yielded two fun-damental observations. First, that se-creted proteins pass through the Golgicomplex on their way out of the cell.This was the first function assigned tothe Golgi complex. Second, secretedproteins never mix with cellular pro-teins in the cytosol; they are segregatedinto vesicles throughout the pathway.These findings were predicated fromtwo important aspects of the experi-mental design. Palades careful use ofelectron microscopy and autoradiogra-phy allowed him to look at the fine de-tails of the pathway. Of equal impor-tance was the choice of a cell typedevoted to secretion, the pancreatic

exocrine cell, as a model system. In adifferent cell type, significant amountsof nonsecreted proteins would havealso been produced during the label-ing, obscuring the fate of secretoryproteins in particular.

Palades work set the stage formore detailed studies. Once the secre-tory pathway was clearly described,entire fields of research were openedup to investigation in the synthesis andmovement of both secreted and mem-brane proteins. For this groundbreak-ing work, Palade was awarded theNobel Prize for Physiology and Medi-cine in 1974.

FIGURE 1 The synthesis and movement of guinea pigpancreatic secretory proteins as revealed by electron microscopeautoradiography. After a period of labeling with [3H]-leucine, thetissue is fixed, sectioned for electron microscopy, and subjected toautoradiography. The radioactive decay of [3H] in newly synthesizedproteins produces autoradiographic grains in an emulsion placed overthe cell section (which appear in the micrograph as dense, wormlikegranules) that mark the position of newly synthesized proteins. (a) At theend of a 3-minute labeling period autoradiographic grains are over therough ER. (b) Following a 7-minute chase period with unlabeled leucine,most of the labeled proteins have moved to the Golgi vesicles. (c) After a37-minute chase, most of the proteins are over immature secretoryvesicles. (d) After a 117-minute chase, the majority of the proteins areover mature zymogen granules. [Courtesy of J. Jamieson and G. Palade.]

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THE INFANCY OF SIGNAL TRANSDUCTIONGTPSTIMULATION OF cAMP SYNTHESIS M. Rodbell et al., 1971, J. Biol. Chem. 246:1877

In the late 1960s the study of hormoneaction blossomed following the discov-ery that cyclic adenosine monophos-phate (cAMP) functioned as a secondmessenger, coupling the hormone-mediated activation of a receptor to acellular response. In setting up an ex-perimental system to investigate thehormone-induced synthesis of cAMP,Martin Rodbell discovered an importantnew player in intracellular signalingguanosine triphosphate (GTP).

Background The discovery of GTPs role in regulat-ing signal transduction began withstudies on how glucagon and otherhormones send a signal across theplasma membrane that eventuallyevokes a cellular response. At the out-set of Rodbells studies, it was knownthat binding of glucagon to specific re-ceptor proteins embedded in the mem-brane stimulates production of cAMP.The formation of cAMP from ATP iscatalyzed by a membrane-bound en-zyme called adenyl cyclase. It had beenproposed that the action of glucagon,and other cAMP-stimulating hor-mones, relied on additional molecularcomponents that couple receptor acti-vation to the production of cAMP.However, in studies with isolated fat-cell membranes known as ghosts,Rodbell and his coworkers were unableto provide any further insight into howglucagon binding leads to an increasein production of cAMP. Rodbell thenbegan a series of studies with a newlydeveloped cell-free system, purified ratliver membranes, which retained bothmembrane-bound and membrane-associated proteins. These experimentseventually led to the finding that GTPis required for the glucagon-inducedstimulation of adenyl cyclase.

The Experiment One of Rodbells first goals was tocharacterize the binding of glucagon tothe glucagon receptor in the cell-freerat liver membrane system. First, puri-fied rat liver membranes were incu-bated with glucagon labeled with theradioactive isotope of iodine (125I).Membranes were then separated fromthe unbound [125I] glucagon by cen-trifugation. Once it was establishedthat labeled glucagon would indeedbind to the purified rat liver cell mem-branes, the study went on to determineif this binding led directly to activationof adenyl cyclase and production ofcAMP in the purified rat liver cellmembranes.

The production of cAMP in thecell-free system required the additionof ATP; the substrate for adenyl cy-clase, Mg2; and an ATP-regeneratingsystem consisting of creatine kinaseand phosphocreatine. Surprisingly,when the glucagon-binding experi-ment was repeated in the presence ofthese additional factors, Rodbell ob-served a 50 percent decrease inglucagon binding. Full binding couldbe restored only when ATP was omit-ted from the reaction. This observationinspired an investigation of the effectof nucleoside triphosphates on thebinding of glucagon to its receptor. Itwas shown that relatively high (i.e.,millimolar) concentrations of not onlyATP but also uridine triphosphate(UTP) and cytidine triphosphate (CTP)reduced the binding of labeledglucagon. In contrast, the reduction ofglucagon binding in the presence ofGTP occurred at far lower (micromo-lar) concentrations. Moreover, lowconcentrations of GTP were found tostimulate the dissociation of boundglucagon from the receptor. Taken to-gether, these studies suggested that

GTP alters the glucagon receptor in amanner that lowers its affinity forglucagon. This decreased affinity bothaffects the ability of glucagon to bindto the receptor and encourages the dis-sociation of bound glucagon.

The observation that GTP was in-volved in the action of glucagon led to asecond key question: Can GTP also ex-ert an affect on adenyl cyclase? Ad-dressing this question experimentallyrequired the addition of both ATP, as asubstrate for adenyl cyclase, and GTP,as the factor being examined, to the pu-rified rat liver membranes. However,the previous study had shown that theconcentration of ATP required as a sub-strate for adenyl cyclase could affectglucagon binding. Might it also stimu-late adenyl cyclase? The concentrationof ATP used in the experiment couldnot be reduced because ATP was readilyhydrolyzed by ATPases present in therat liver membrane. To get around thisdilemma, Rodbell replaced ATP with anAMP analog, 5-adenyl-imidodiphos-phate (AMP-PNP), which can be con-verted to cAMP by adenyl cyclase, yet isresistant to hydrolysis by membraneATPases. The critical experiment nowcould be performed. Purified rat livermembranes were treated with glucagonboth in the presence and absence ofGTP, and the production of cAMP fromAMP-PNP was measured. The additionof GTP clearly stimulated the produc-tion of cAMP when compared toglucagon alone (Figure 1) indicatingthat GTP affects not only the binding ofglucagon to its receptor but also stimu-lates the activation of adenylyl cyclase.

Discussion Two key factors led Rodbell and hiscolleagues to detect the role of GTP insignal transduction, whereas previous


studies had failed to do so. First, byswitching from fat-cell ghosts to the ratliver membrane system, the Rodbell re-searchers avoided contamination of theircell-free system with GTP, a problem as-sociated with the procedure for isolatingghosts. Such contamination would maskthe effects of GTP on glucagon bindingand activation of adenyl cyclase. Second,when ATP was first shown to influenceglucagon binding, Rodbell did not sim-ply accept the plausible explanation thatATP, the substrate for adenyl cyclase,also affects binding of glucagon. Instead,he chose to test the effects on binding ofthe other common nucleoside triphos-phates. Rodbell later noted that he knewcommercial preparations of ATP often

are contaminated with low concentra-tions of other nucleoside triphosphates.The possibility of contamination sug-gested to him that small concentrationsof GTP might exert large effects onglucagon binding and the stimulation ofadenyl cyclase.

This critical series of experimentsstimulated a large number of studieson the role of GTP in hormone action,eventually leading to the discovery ofG proteins, the GTP-binding proteinsthat couple certain receptors to theadenyl cyclase. Subsequently, an enor-mous family of receptors that requireG proteins to transduce their signalswere identified in eukaryotes from yeastto humans. These G protein-coupled

receptors are involved in the action ofmany hormones as well as in a numberof other biological activities, includingneurotransmission and the immune re-sponse. It is now known that bindingof ligands to their cognate G protein-coupled receptors stimulates the asso-ciated G proteins to bind GTP. Thisbinding causes transduction of a signalthat stimulates adenyl cyclase to pro-duce cAMP and also desensitization ofthe receptor, which then releases itsligand. Both of these affects were ob-served in Rodbells experiments onglucagon action. For these seminal ob-servations, Rodbell was awarded theNobel Prize for Physiology and Medi-cine in 1994.

FIGURE 1 Effect of GTP on glucagon-stimulated cAMPproduction from AMP-PNP by purified rat liver membranes. Inthe absence of GTP, glucagon stimulates cAMP formation abouttwofold over the basal level in the absence of added hormone. WhenGTP also is added, cAMP production increases another fivefold.[Adapted from M. Rodbell et al., 1971, J. Biol. Chem. 246:1877.]

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SENDING A SIGNAL THROUGH A GASM. T. Kahn and R. Furchgott, 1987, in M. J. Rand and C. Raper, eds., Pharmacology, Elsevier Science Publisher, pp. 341344;R. M. J. Palmer et al., 1987, Nature, 327:524; and L. J. Ignarro et al., Proc. Natl. Acad. Sci. USA 84:9265

For decades scientists have tried to un-derstand how cells work together intissues, as well as in whole organisms.By the 1980s, the identity of many sig-naling molecules, the cellular responsesthey evoked, and many aspects ofintracellular signaling pathways wereunderstood. All the known signalingmoleculesthe familiar hormones andneurotransmitterswere nongaseoussubstances, primarily peptides andamino acid derivatives. However, stud-ies on the dilation of blood vesselsshowed that the gas nitric oxide (NO)could indeed function as a signalingmolecule.

Background The discovery of nitric oxide as a sig-naling molecule began with studies onthe mechanism by which blood vesselsrelax and constrict, processes knownas vasodilation and vasoconstriction.In addition to their desire to understandthe basic biology of these processes,scientists recognized its medical impor-tance, as drugs that promote vasodila-tion could aid in the treatment ofcardiovascular diseases. Nitroglycerin,long used to treat angina pectoris, wasknown to promote vasodilation. Whenapplied to isolated blood vessels, nitro-glycerin and other nitrogen-containingcompounds had been found to activatea signaling pathway that began bystimulating the production of cyclicguanosine monophosphate (cGMP),and eventually resulted in dilation. Therewas much interest in discovering thenatural sig

classic experiments in molecular biology

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