INTRODUCTION to MOLECULAR BIOLOGY 4TH PAGES © Jones & Bartlett Learning, LLC

Introduction© Jones & Bartlett Learning, LLCto © Jones & Bartlett Learning, LLC MolecularNOT FOR SALE OR DISTRIBUTION Biology NOT FOR SALE OR DISTRIBUTION

CHAPTER OUTLINE © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR1.1 SALE Intellectual OR DISTRIBUTION Foundation NOT FOR1.5 SALEWatson-Crick OR DISTRIBUTION Model Two studies performed in the 1860s provided the intellectual Rosalind Franklin and Maurice Wilkins obtained x-ray diffraction underpinning for molecular biology. patterns of extended DNA fibers. James Watson and Francis Crick proposed that DNA is a double- 1.2 One Gene–One Polypeptide Hypothesis stranded helix. Each gene is responsible© Jones for the synthesis & Bartlett of a single Learning, polypeptide. LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION1.6 Central Dogma NOT FOR SALE OR DISTRIBUTION 1.3 Introduction to Nucleic Acids The central dogma provides the theoretical framework for molecular DNA contains the sugar deoxyribose and RNA contains the sugar biology. ribose. A nucleoside is formed by attaching a purine or pyrimidine base to a 1.7 Introduction to Recombinant DNA ©sugar. Jones & Bartlett Learning, LLC Technology© Jones & Bartlett Learning, LLC NOTA nucleotide FOR is formedSALE by attachingOR DISTRIBUTION a phosphate group to the sugar RecombinantNOT FOR DNA technology SALE ORallows DISTRIBUTION us to study complex biological group in a nucleoside. systems. DNA is a linear chain of deoxyribonucleotides. A great deal of molecular biology information is available on the Internet. 1.4 DNA: Hereditary Material Transformation experiments led to the discovery that DNA is the QUESTIONS AND PROBLEMS © Jones & Bartletthereditary Learning, material. LLC © Jones & Bartlett Learning, LLC NOT FOR SALEChemical OR experiments DISTRIBUTION also supported the hypothesis that DNA NOTis the FORSUGGESTED SALE OR READING DISTRIBUTION hereditary material.

1 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION he firstNOT recorded FOR useSALE of the OR term DISTRIBUTION molecular biology appears to have been in a 1938 report prepared for the Rockefeller Foun- Tdation by Warren Weaver, the director of the Foundation’s Natural Science Division. The report proposed that the Foundation should start to fund research efforts to seek molecular explanations © Jones & Bartlettfor biologicalLearning, processes. LLC Weaver used the ©term Jones “molecular & Bartlett biology” Learning, LLC NOT FOR SALEto OR describe DISTRIBUTION a research approach he envisionedNOT would FOR use SALE the physical OR DISTRIBUTION sciences to address fundamental biological problems. Weaver’s pro- posal was remarkably farsighted, especially when one considers many of his contemporaries believed living cells possessed a vital force that © Jones & Bartlett Learning,could LLC not be explained by chemical© Jones or & physical Bartlett laws Learning, that govern LLC the NOT FOR SALE OR DISTRIBUTIONinanimate world. NOT FOR SALE OR DISTRIBUTION At the time of Weaver’s report two new disciplines—genetics and biochemistry—were altering the way biologists thought about living systems. Geneticists established that the functional and physical unit of heredity is the gene. However, they did not know the chemical © Jones & Bartlett Learning, LLC nature of genes,© Jones the way & Bartlett that hereditary Learning, information LLC is stored in NOT FOR SALE OR DISTRIBUTION genes, how NOTgenes FORare replicated SALE OR so DISTRIBUTIONthey can be transmitted to the next generation, or how information stored in genes determines a specific physical trait such as eye color. Biochemists had delivered a major blow to the vital force theory by demonstrating that cell- © Jones & Bartlettfree extractsLearning, can LLC perform many of the same© functionsJones & as Bartlett intact cells. Learning, LLC NOT FOR SALEHowever, OR DISTRIBUTION they knew very little about proteinNOT or nucleic FOR SALEacid structure OR DISTRIBUTION and function. Neither genetics nor biochemistry had the power to investigate the chemical basis of heredity on its own. In fact, it took an interdisciplinary effort involving specialists in many fields of the life sciences, includ- © Jones & Bartlett Learning,ing LLC genetics, biochemistry, biophysics,© Jones µbiology, Bartlett Learning, chemistry, LLC x-ray NOT FOR SALE OR DISTRIBUTIONcrystallography, virology, developmentalNOT FOR SALE biology, OR andDISTRIBUTION immunology, to solve the hereditary problem. This interdisciplinary effort resulted in the creation of a new discipline, molecular biology, which has had a remarkable impact on all areas of the life sciences from agronomy © Jones & Bartlett Learning, LLC to zoology. The© Jones scope of& thisBartlett book isLearning, limited to anLLC examination of the NOT FOR SALE OR DISTRIBUTION contributionsNOT that FOR molecular SALE biology OR DISTRIBUTION has made to understanding the flow of information from genes to proteins.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE1. OR1 DISTRIBUTIONIntellectual Foundation NOT FOR SALE OR DISTRIBUTION Two studies performed in the 1860s provided the intellectual underpinning for molecular biology. © Jones & Bartlett Learning,The LLC earliest intellectual roots© ofJones molecular & Bartlett biology Learning,can be traced LLC back NOT FOR SALE OR DISTRIBUTIONto the work of two investigatorsNOT FOR in the SALE 1860s. OR No DISTRIBUTION connection was apparent between the experiments performed by the two investigators for more than 75 years, but when the connection finally was made, the result was the birth of molecular biology and the beginning of a scientific revolution that continues today. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

2 CHAPTER 1 INTRODUCTION TO MOLECULAR BIOLOGY 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALEThe workOR DISTRIBUTION of the first investigator, Gregor Mendel,NOT FORan Austrian SALE OR DISTRIBUTION monk and botanist, is familiar to all biology students and so is only summarized briefly here. Mendel discovered three basic laws of inheritance by studying the way in which simple physical traits are passed on from one generation of pea plants to the next. For convenience, Mendel’s laws of inheritance© Jones &will Bartlett be described Learning, using LLC two modern © Jones & Bartlett Learning, LLC biological terms, geneNOT for FOR a unit SALE of heredity OR DISTRIBUTION and chromosome for a NOT FOR SALE OR DISTRIBUTION structure bearing several linked genes. 1. The law of segregation: A specific gene may exist in alternate © Jonesforms called & Bartlett alleles .Learning, An organism LLC inherits one allele for each© Jones & Bartlett Learning, LLC trait from each parent. The two alleles, which may be the same NOTor different,FOR SALE segregate OR DISTRIBUTION (or separate) during germ cell (spermNOT or FOR SALE OR DISTRIBUTION egg) formation and combine again as a result of fertilization so that each parent transmits one allele to each offspring. 2. The law of independent assortment: Specific physical traits such © Jones & Bartlettas plant Learning, size and color LLC are inherited independently© Jones of one another.& Bartlett Learning, LLC NOT FOR SALEMendel OR DISTRIBUTION was fortunate to have selected physicalNOT traits FOR that SALE were OR DISTRIBUTION determined by genes that were on different chromosomes. 3. The law of dominance: An allele may be dominant or recessive. A dominant allele produces its characteristic physical trait whether it is paired with an identical allele or a recessive allele. In con- © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC trast, a recessive allele produces its characteristic physical trait only when pairedNOT with FOR an SALE identical OR allele. DISTRIBUTION In pea plants, tallness NOT FOR SALE OR DISTRIBUTION is dominant and shortness recessive. When Mendel allowed pea plants with one allele for tallness and one allele for shortness to self-fertilize, he observed three times as many progeny were © Jonestall as short. & Bartlett Today, Learning,we know there LLC are exceptions to the law© of Jones & Bartlett Learning, LLC NOTdominance. FOR SALE Sometimes OR DISTRIBUTION neither allele is dominant. For instance,NOT a FOR SALE OR DISTRIBUTION plant that inherits a gene for a red flower and a gene for a white flower may produce an intermediate phenotype, a pink flower. Unfortunately, scientists failed to recognize the significance of © Jones &Mendel’s Bartlett work Learning, during his LLC lifetime. His paper remained© Jones little-known & Bartlett Learning, LLC NOT FORuntil SALE about OR 1900, DISTRIBUTION when scientists rediscovered Mendel’sNOT laws FOR of inheriSALE- OR DISTRIBUTION tance, giving birth to the science of genetics. The second investigator, the Swiss physician Friedrich Miescher, performed experiments that led to the discovery of deoxyribonucleic acid (DNA), which we, of course, now know is the hereditary material. Miescher did not set ©out Jones to discover & Bartlett the hereditary Learning, material LLC but instead © Jones & Bartlett Learning, LLC was interested in studyingNOT cell FOR nuclei SALE from whiteOR DISTRIBUTION blood cells, which he col- NOT FOR SALE OR DISTRIBUTION lected from pus discharges on discarded bandages used to cover infected wounds. Miescher used a combination of protease (an enzyme that hydro- lyzes proteins) digestion and solvent extraction to disrupt and fractionate the white© Jones blood & cells. Bartlett One fraction, Learning, which LLC he called nuclein, contained© Jones & Bartlett Learning, LLC an acidicNOT materialFOR SALE with unusuallyOR DISTRIBUTION high phosphorus content. MiescherNOT FOR SALE OR DISTRIBUTION later found that salmon sperm cells, which have remarkably large cell nuclei, are also an excellent source of nuclein. In 1889 Miescher’s student, Richard Altmann, separated nuclein into protein and a substance with a very high phosphorous content that he named nucleic acid. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

1.1 Intellectual Foundation 3 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION 1.2 OneNOT Gene–One FOR SALE Polypeptide OR DISTRIBUTION Hypothesis Each gene is responsible for the synthesis of a single polypeptide. © Jones & BartlettMendel’s Learning, experiments LLC showed that the genetic© Jonesmakeup &of Bartlettan organism, Learning, LLC NOT FOR SALEits OR genotype DISTRIBUTION, determines the organism’s physicalNOT traits,FOR SALEits phenotype OR DISTRIBUTION. However, his experiments did not show how genes are able to deter- mine complex physical traits such as plant color or size. Archibald Gar- rod, an English physician, was the first to provide an explanation for the relationship between genotype and phenotype. Garrod uncovered © Jones & Bartlett Learning,this LLC relationship in the early ©1920s Jones while & studyingBartlett alkaptonuria, Learning, LLC a rare NOT FOR SALE OR DISTRIBUTIONinherited human disorder inNOT which FOR the SALEurine of OR affected DISTRIBUTION individuals becomes very dark upon standing due to the oxidation of homogentisic acid, a breakdown product of the amino acid tyrosine. Garrod cor- rectly proposed that alkaptonuria results from a recessive gene, which © Jones & Bartlett Learning, LLC causes a deficiency© Jones in an & enzyme Bartlett needed Learning, to convert LLC homogentisic acid to colorless products. NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Garrod’s work was generally ignored until the early 1940s, when two American scientists, George Beadle and Edward Tatum, provided additional experimental proof for a relationship between genes and enzymes. Beadle and Tatum thought that if a gene really does specify © Jones & Bartlettan enzyme, Learning, it should LLC be possible to create genetic© Jones mutants & Bartlett that cannot Learning, LLC NOT FOR SALEcarry OR outDISTRIBUTION specific enzymatic reactions in a biochemicalNOT FOR pathway. SALE OR They DISTRIBUTION decided to work with the bread mold, Neurospora crassa, because it could be subjected to genetic analysis. In addition, N. crassa has very simple nutritional requirements. It can grow in a minimal medium that © Jones & Bartlett Learning,contains LLC a single carbon source© Jones such as& sucrose,Bartlett inorganic Learning, salts, LLC and the vitamin biotin. Beadle and Tatum began by irradiating N. crassa NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION with x-rays to generate mutants with additional nutritional growth requirements. A mutant that requires a specific nutritional supplement that is not required by the parent cell is called an auxotroph. In one set of experiments Beadle and Tatum focused particular © Jones & Bartlett Learning, LLC attention on ©arginine Jones auxotrophs. & Bartlett Genetic Learning, analysis LLC of these auxotrophs revealed that mutations in three different genes produce auxotrophs NOT FOR HOSALE OR DISTRIBUTIONHO NOT FOR SALE OR DISTRIBUTION for the amino acid arginine. Each kind of auxotroph was blocked at C C 1 1 a specific step in the arginine biosynthetic pathway and accumulated H 2 OHC H 2 HC large quantities of the substance formed just before the blocked step. Beadle and Tatum thus had replicated in the bread mold the same HO3 HC HO3 HC © Jones & Bartletttype Learning,of situation LLC Garrod had observed in ©alkaptonuria. Jones & Bartlett A defective Learning, LLC HO4 HC NOTHO 4FORHC SALEgene OR caused DISTRIBUTION a defect in a specific enzyme thatNOT resulted FOR in SALE the abnormal OR DISTRIBUTION

C5H2OH C5H2OH accumulation of an intermediate in a metabolic pathway. As a result of their work with the N. crassa mutants, Beadle and (a) Ribose (b) Deoxyribose Tatum proposed the one gene–one enzyme hypothesis, which states FIGURE 1.1 ©The Jones two sugars & Bartlett present inLearning, nucleic that LLC each gene is responsible© Jones for synthesizing & Bartlett a singleLearning, enzyme. LLC We acids. (a) RiboseNOT and FOR (b) deoxyriboseSALE OR each DISTRIBUTION now know that many enzymesNOT areFOR made SALE of more OR DISTRIBUTIONthan one type of contain five carbon atoms and an aldehyde polypeptide chain and that a single mutation may affect just one of group and therefore belong to aldopentose the polypeptide chains. The original one gene–one enzyme hypothesis family of simple sugars. The only difference between the two sugars is that ribose has a hence was modified to become a one gene–one polypeptide hypoth- hydroxyl group at carbon-2 and deoxyribose esis. As we will see later, however, even the one gene–one polypeptide © Jonesas a hydrogen & Bartlett atom Learning,at carbon-2. LLC hypothesis is© an Jones oversimplification. & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

4 CHAPTER 1 INTRODUCTION TO MOLECULAR BIOLOGY 3RD PAGES

Tropp Molecular Biology of Genes Jones and Bartlett Publishers Elizabeth Morales Illustration Services Image 09166X_Ch01_Fig01.eps Date 01-15-07 © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

5 CH2OH 5 OH 5 H H HOH2C H HOH2C O 4 O 4 O 4 CHOH HH 1 HH 1 HH 1 OH Rotate about H H 3 2 © Jones & Bartlett3 2 Learning, LLC 3 2 © Jones & Bartlett Learning, LLC C3-C4 bond OH OH NOT FOR SALEOH OH OR DISTRIBUTION OHOH NOT FOR SALE OR DISTRIBUTION

(b) Cyclization to form deoxyribofuranose 5 5 OH 5 CH2OH HOH2C O H H HOH2C H 4 CHOH 4 O HH 1 4 © Jones &O Bartlett Learning,HH LLC1 © Jones & Bartlett Learning, LLC HH 1 H H 3 2 OH NOT FOR SALE OR DISTRIBUTION3 2 NOT FOR SALE OR DISTRIBUTION 3 2 Rotate about C3-C4 bond OH H OH H OH H

HC CH FIGURE 1.2 Conversion of straight chain ribose and deoxyribose to cyclic forms. (a) Conversion of ribose to ribofuranose and (b) conversion of deoxyribose to deoxyribofuranose.© Jones & (c) Bartlett Structure Learning, of furan, a five-atom LLC ring © Jones & Bartlett Learning, LLC system. NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

CH2OH CH2OH 1.3© IntroductionJones & Bartlett to Learning, Nucleic LLCAcids © Jones & BartlettO Learning, LLCO OH NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION DNA contains the sugar deoxyribose and RNA contains the OH sugar ribose. OHOH OHOH Investigators slowly came to realize that nucleic acids could be divided α-D-ribofuranose β-D-ribofuranose © Jones &into Bartlett two major Learning, groups: DNALLC and ribonucleic acid© (RNA). Jones The & Bartlettprin- Learning, LLC NOT FORcipal SALE difference OR DISTRIBUTION between DNA and RNA is that theNOT former FOR contains SALE OR DISTRIBUTION deoxyribose and the latter contains ribose (FIGURE 1.1). The two five- CH2OH CH2OH OH carbon sugars (pentoses) differ in only one substituent: deoxyribose O O has a hydrogen atom at carbon-2, whereas ribose has a hydroxyl group at this position. By convention, the aldehyde group in the pentose is OH C-1, the next carbon© is Jones C-2, and & Bartlettso forth. Learning, LLC OH© Jones & BartlettOH Learning, LLC Each pentose chainNOT can FOR close SALE to form OR a five-carbon DISTRIBUTION ring in which α-D-deoxyribofuranoseNOT FOR SALEβ-D-deoxyribofuranose OR DISTRIBUTION an oxygen bridge joins C-1 to C-4 (FIGURE 1.2ab). Because the sugar FIGURE 1.3 Haworth structures for rings are derivatives of furan (Figure 1.2c) they are called furanoses. ribofuranose and deoxyribofuranose. Haworth Ribofuranose and deoxyribofuranose are often depicted as Haworth structure represents a cyclic sugar as a structures© Jones (named & Bartlett after Walter Learning, N. Haworth, LLC the investigator who© Jones flat ring & Bartlettperpendicular Learning, to the plane LLC of the devisedNOT the FOR representations). SALE OR DISTRIBUTION As illustrated in FIGURE 1.3, a HaworthNOT pageFOR with SALE the ring-oxygen OR DISTRIBUTION in the back and structure represents a cyclic sugar as a flat ring perpendicular to the C-1 to the right. A hydrogen atom attached plane of the page with the ring-oxygen in the back and C-1 to the to the sugar ring is represented by a line. Ring formation can lead to two different right. The ring’s thick lower edge projects toward the viewer, its upper stereochemical arrangements at C-1: one in edge projects back behind the page, and its substituents are visualized which the hydroxyl at C-1 points down and © Jones &as Bartlettbeing either Learning, above or LLCbelow the plane of the ring.© AJones line is &used Bartlett to another Learning, in which LLC it points up. NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

1.3 Introduction to Nucleic Acids 5 4TH PAGES

Tropp Molecular Biology of Genes Jones and Bartlett Publishers Elizabeth Morales Illustration Services Image 09166-X_Ch01_Fig03.eps Date 01-15-07 © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION (a) NOT FOR SALE OR DISTRIBUTION O NH2 4 4 4 3 3 CH 3 5 3 5 N HN 5 N 2 2 2 6 6 6 O © Jones & Bartlett Learning,1 N LLC O 1 N 1©N Jones & Bartlett Learning, LLC H H NOT FOR SALE OR DISTRIBUTIONThymine (T) CytosineNOT (C) FOR SALE OR DISTRIBUTION Pyrimidine Pyrimidine bases

(b) NH2 O 6 6 7 6 7 7 © Jones & Bartlett Learning, LLC 5 N © Jones5 N & Bartlett Learning,5 N LLC N 1 N HN 1 1 NOT FOR SALE OR DISTRIBUTION 8 NOT FOR SALE8 OR DISTRIBUTION8 2 2 2 4 N 9 4 N 9 4 N 9 3 N 3 N H N 3 N H H 2 H

Adenine (A) Guanine (G) © Jones & Bartlett Learning, LLC ©Purine Jones & Bartlett Learning, Purine bases LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION O FIGURE 1.4 Pyrimidine and purine bases in DNA. (a) Pyrimidine bases and 4 (b) purine bases. 3 HN 5 2 6 O 1 N © Jones & Bartlettrepresent Learning, a hydrogen LLC atom attached to the sugar© Jones ring. &Ring Bartlett formation Learning, LLC H NOT FOR SALEcan OR lead DISTRIBUTION to two different stereochemical arrangementsNOT FOR SALE at C-1. OR One DISTRIBUTION Uracil (U) arrangement, the a-anomer, is represented by drawing the hydroxyl FIGURE 1.5 Uracil (U). group attached to C-1 below the plane of the ring and the other, the b-anomer, by drawing it above the plane of the ring. (Although ribofuranose and deoxyribofuranose actually have puckered rather than O NH2 © Jones4 & Bartlett Learning,4 planar LLC conformations, Haworth© Jones structures & Bartlett are convenient Learning, representa LLC - 3 CH 3 3 5 HNNOT FOR5 SALE ORN DISTRIBUTIONtions for the pentose rings whenNOT precise FOR three-dimensionalSALE OR DISTRIBUTION information 2 2 6 6 is not required and, therefore, are used throughout this book.) O 1 N O 1 N 5′CH2OH 5′CH2OH O O A nucleoside is formed by attaching a purine or pyrimidine base to © Jones4′ & Bartlett1′ Learning,4′ LLC 1′ a sugar. © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 3′ 2′ 3′ 2′ Each deoxyribose group in DNA is attached to one of four different OH OH nitrogen heterocyclic molecules, referred to as bases because they can Deoxythymidine Deoxycytidine act as proton acceptors. Two bases, thymine (T) and cytosine (C), are Tropp Molecular Biology of Genes derivatives of pyrimidine (FIGURE 1.4a) and the other two, adenine (A) Jones and Bartlett Publishers Elizabeth Morales Illustration© Jones Services & Bartlettand Learning,guanine (G) ,LLC are derivatives of purine© (Figure Jones 1.4b). & Bartlett RNA also Learning, LLC NH Image2 09166-X_Ch01_Fig04.epsNOT O FOR Date SALE 01-22-07contains OR DISTRIBUTION cytosine, adenine, and guanine, NOTbut the FOR pyrimidine SALE ORuracil DISTRIBUTION 6 6 7 7 5 N 5 N FIGURE 1.5 1 N HN (U) replaces thymine ( ). The only difference between uracil 1 8 8 and thymine is that the latter contains a methyl group attached to 2 2 4 N 9 4 N 9 carbon-5. T, C, A, and G combine with deoxyribose to form a class 3 N H2N 3 N of compounds called deoxyribonucleosides (FIGURE 1.6), and U, C, 5′CH OH © Jones & Bartlett5′CH OH Learning, LLC © Jones & Bartlett Learning, LLC 2 2 A, and G combine with ribose to form a related class of compounds O NOT FOR SALE ORO DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 4′ 1′ 4′ 1′ called ribonucleosides (FIGURE 1.7). Each base is linked to the pentose ring by a bond that joins a specific nitrogen atom on the base (N-1 in 3′ 2′ 3′ 2′ pyrimidines and N-9 in purines) to C-1 on the furanose ring. This bond OH OH is termed an N-glycosylic bond. Because each nucleoside has two ring Deoxyadenosine Deoxyguanosine © Jones & Bartlett Learning, LLC systems (the ©sugar Jones and the& Bartlett base attached Learning, to it), a LLC method is required to

NOTFIGURE TroppFOR Molecular 1.SALE6 Deoxyribonucleosides. Biology OR DISTRIBUTION of Genes distinguish betweenNOT FOR atoms SALE in each OR ring DISTRIBUTION system. This problem is solved Jones and Bartlett Publishers Elizabeth6 Morales Illustration ServicesCHAPTER 1 INTRODUCTION TO MOLECULAR BIOLOGY Image 09166-X_Ch01_Fig05.eps Date 01-02-07 4TH PAGES

Tropp Molecular Biology of Genes Jones and Bartlett Publishers Elizabeth Morales Illustration Services Image 09166-X_Ch01_Fig06.eps Date 01-15-07 © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC O NOT FORby SALE adding OR a prime DISTRIBUTION (′) after the sugar atoms. The firstNOT carbon FOR atom SALE in OR DISTRIBUTION NH2 4 4 the sugar thus becomes 1′, the second 2′, and so forth. 3 3 5 HN 5 N 2 2 6 6 O A nucleotide is formed by attaching a phosphate group to the 1 N O 1 N sugar group in a nucleoside.© Jones & Bartlett Learning, LLC 5′CH2OH © Jones & Bartlett5′CH2OH Learning, LLC O O Nucleosides that haveNOT a phosphate FOR SALE group OR attached DISTRIBUTION to the sugar group NOT FOR SALE OR DISTRIBUTION 4′ 1′ 4′ 1′ are called nucleotides. Ribonucleoside and deoxyribonucleoside derivatives are called ribonucleotides and deoxyribonucleotides, respectively 3′ 2′ 3′ 2′ OH OH OH OH (FIGURE 1.8). The pentose carbon atom to which the phosphate group is attached© Jones is given & Bartlett as part ofLearning, the nucleotide’s LLC name. Thus, the phos© -Jones &Uridine Bartlett Learning, LLCCytidine phateNOT group FOR is attached SALE to OR C-5 DISTRIBUTION′ in uridine-5′-monophosphate (5′-UMP)NOT FOR SALE OR DISTRIBUTION and thymidine-5′-monophosphate (5′-dTMP) and to C-3′ in uridine- NH2 O 3 -monophosphate (3 -UMP) and thymidine-3 -monophosphate (3 - 6 ′ ′ ′ ′ 6 7 7 5 N 5 N dTMP). As indicated in TABLE 1.1, nucleoside monophosphates have two 1 N HN 1 alternative names. For example, cytidine-5 -monophosphate (5 -CMP) 8 8 ′ ′ 2 2 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,4 N LLC9 4 9 is also known as 5′-cytidylate. Each nucleoside monophosphate is 3 N H N 3 N N NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 2 5′CH2OH 5′CH2OH O O 4′ 1′ 4′ 1′ (a) 5′-nucleoside monophosphates O O 3′ 2′ 3′ 2′ 4 4 ©3 Jones & Bartlett Learning,3 LLC CH OH© OH Jones & BartlettOH Learning, OH LLC HN HN 3 NOT FOR5 SALE OR DISTRIBUTION5 AdenosineNOT FOR SALE GuanosineOR DISTRIBUTION 2 2 6 6 O O 1 N 1 N FIGURE 1.7 Ribonucleosides. – – HO3POCH2 HO3POCH2 5′ O 5′ O © Jones4′ & Bartlett1′ Learning, LLC4′ 1′ © Jones & Bartlett Learning, LLC

NOT FOR3′ SALE2′ OR DISTRIBUTION3′ 2′ NOT FOR SALE OR DISTRIBUTION OH OH OH Uridine-5′-monophosphate (5′-UMP) Thymidine-5′-monophosphate (5′-dTMP) or 5′-uridylate or 5′-thymidylate

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC (b) 3′-nucleoside monophosphates NOT FOR SALE OR DISTRIBUTIONO NOT FORO SALE OR DISTRIBUTION 4 4 3 3 CH3 HN 5 HN 5 2 2 6 6 O O ©1 JonesN & Bartlett Learning, 1LLCN © Jones & Bartlett Learning, LLC CH2OH CH2OH 5′ O NOT FOR SALE OR5 ′DISTRIBUTIONO NOT FOR SALE OR DISTRIBUTION 4′ 1′ 4′ 1′ Tropp Molecular Biology of Genes 3′ 2′ 3′ 2′ Jones and Bartlett Publishers OH – Elizabeth Morales Illustration Services – OPO3H © Jones OPO& Bartlett3H Learning, LLC © JonesImage & 09166-X_Ch01_Fig07.eps Bartlett Learning, DateLLC 01-15-07 NOTUridine-3 FOR′-monophosphate SALE OR (3′-UMP) DISTRIBUTIONThymidine-3′-monophosphate (3′-dTMP)NOT FOR SALE OR DISTRIBUTION or 3′-uridylate or 3′-thymidylate

FIGURE 1.8 Nucleotides. Nucleotides are formed by adding a phosphate group to the pentose ring in a nucleoside. (a) Nucleotides formed by adding a phosphate group to the 5′-hydroxyl group in uridine or thymidine. (b) © Jones &Nucleotides Bartlett formed Learning, by adding LLC a phosphate group to the© 3 ′-hydroxylJones & group Bartlett Learning, LLC NOT FORin SALE uridine ORor thymidine. DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

1.3 Introduction to Nucleic Acids 7 4TH PAGES

Tropp Molecular Biology of Genes Jones and Bartlett Publishers Elizabeth Morales Illustration Services Image 09166-X_Ch01_Fig08.eps Date 01-15-07 © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION TABLE 1.1 NOTBases, FOR Nucleosides, SALE OR and DISTRIBUTION Nucleotides Base Sugar Nucleoside 5’-Mononucleotide

Uracil (U) ribose uridine Uridine-5’-monophosphate or © Jones & Bartlett Learning, LLC 5’-uridylate© Jones (5’-UMP) & Bartlett Learning, LLC NOT FOR SALE CytosineOR DISTRIBUTION (C) ribose cytidine Cytidine-5’-monophosphateNOT FOR SALE OR or DISTRIBUTION 5’-cytidylate (5’-CMP) Adenine (A) ribose adenosine Adenosine-5’-monophosphate or 5’-adenylate (5’-AMP) Guanine (G) ribose guanosine Guanosine-5’-monophosphate or © Jones & Bartlett Learning, LLC © Jones & 5’-guanylateBartlett Learning, (5’-GMP) LLC NOT FOR SALE OR DISTRIBUTIONThymine (T) deoxyribose deoxythymidineNOT FOR1 SALEDeoxythymidine-5’-monophos OR DISTRIBUTION- phate or 5’-deoxythymidylate (5’-dTMP)1 Cytosine (C) deoxyribose deoxycytidine Deoxycytidine-5’-monophosphate or 5’-deoxycytidylate © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,(5’-dCMP) LLC NOT FOR SALE OR DISTRIBUTION Adenine (A) NOTdeoxyribose FOR SALEdeoxyadenosine OR DISTRIBUTIONDeoxyadenosine-5’-monophosphate or 5’-deoxyadenylate (5’-dAMP) Guanine (G) deoxyribose deoxyguanosine Deoxyguanosine-5’-monophosphate or 5’-deoxyguanylate © Jones & Bartlett Learning, LLC (5’-dGMP)© Jones & Bartlett Learning, LLC 1Deoxythymidine and deoxythymidine-5’-monophosphate are also called thymidine and NOT FOR SALE thymidine-5’-monophosphate,OR DISTRIBUTION respectively. When thymineNOT is attached FOR to SALE ribose, theOR DISTRIBUTION nucleoside is called ribothymidine and the nucleotide is called ribothymidylate. This nomenclature convention follows from the fact that thymine is most frequently attached to deoxyribose.

phosphomonoester because it has a single sugar group attached to a phosphate by an ester bond. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION DNA is a linearNOT chain FOR of SALE deoxyribonucleotides. OR DISTRIBUTION The deoxyribonucleotide groups in DNA form a linear polymer in which phosphate groups join 5′- and 3′-carbons of neighboring deoxyribonucleosides (FIGURE 1.9a). The bond that joins neighboring © Jones & Bartlettnucleosides Learning, to a LLCcommon phosphate group© is Jones called &a phosphodiesBartlett Learning,- LLC NOT FOR SALEter OR bond DISTRIBUTION. Each linear DNA chain has a 5′NOT- and FORa 3′-terminus SALE OR. This DISTRIBUTION directionality will be very important in future discussions of DNA structure and function. The structure of RNA is very similar to that for DNA (FIGURE 1.10a). © Jones & Bartlett Learning, LLCJust 3 years after the discovery© Jones that & nucleic Bartlett acids Learning, are linear LLCchains of nucleotides, Frederick Sanger, working in England, showed that NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION each kind of polypeptide molecule is a linear chain of amino acids arranged in a specific order. The amino acid order is responsible for the unique properties of each type of protein, including its ability to catalyze specific reactions, protect cells from foreign organisms and © Jones & Bartlett Learning, LLC substances, ©transport Jones &materials, Bartlett or Learning, support the LLC cellular infrastruc- NOT FOR SALE OR DISTRIBUTION ture. The awarenessNOT FOR that SALE DNA molecules OR DISTRIBUTION are made of linear chains of

8 CHAPTER 1 INTRODUCTION TO MOLECULAR BIOLOGY 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR(a) SALE OR DISTRIBUTION (b) NOT FOR(c) SALE OR DISTRIBUTION 5′ end pApCpGpTp P O 5′ A 3′ P Na+ – O P O 5′ C © JonesNH2 & Bartlett Learning,3′ LLC © Jones & Bartlett Learning, LLC O NOT FOR SALE OR DISTRIBUTIONP NOT FOR SALE OR DISTRIBUTION N 5′ G N 3′ A P T 5 5′ ′CH2 N N 3′ O P © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR3′ SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Na+ – O P O NH2 O © Jones & Bartlett Learning, LLCN © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONC NOT FOR SALE OR DISTRIBUTION O 5′CH2 N O

3′ © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC

O NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Na+ – O P O O O N NH © Jones & Bartlett Learning,G LLC © Jones & Bartlett Learning, LLC NOT FOR5′CH SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 2 N N NH2 O

3′ © Jones & Bartlett Learning,O LLC © Jones & Bartlett Learning, LLC

NOT FOR SALE ORNa DISTRIBUTION+ – O P O NOT FOR SALE OR DISTRIBUTION O O H C 3 NH T

5′CH© 2Jones &N BartlettO Learning, LLC © Jones & Bartlett Learning, LLC NOTO FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

3′

3′ end © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Tropp Molecular Biology of Genes FIGURE 1.9 Segment of a polydeoxyribonucleotide. (a) Extended structure Jones and Bartlett Publishers NOT FORas SALE a sodium OR salt, DISTRIBUTION (b) stick figure structure, and (c) an abbreviatedNOT FOR structure. SALE OR DISTRIBUTION Elizabeth Morales Illustration Services Image 09166-X_Ch01_Fig10.eps Date 02-01-07 1.3 Introduction to Nucleic Acids 9 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION (a) NOT FOR SALE OR DISTRIBUTION(b) (c) 5′ end pApCpGpUp P OH O 5′ A 3′ P OH Na+ – O P O 5′ C © Jones & Bartlett Learning, LLC NH2 © Jones3′ & Bartlett Learning, LLC O P OH NOT FOR SALE OR DISTRIBUTIONN 5′ NOT FOR GSALE OR DISTRIBUTION N 3′ A P OH 5′ U 5′CH2 N N 3′ O P © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION 3′ NOT FOR SALE OR DISTRIBUTION OH O

Na+ – O P O NH2 O © Jones & Bartlett Learning, LLC © Jones & BartlettN Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FORC SALE OR DISTRIBUTION O 5′ CH2 N O

Na+ – O P O O O N NH © Jones & Bartlett Learning, LLC © JonesG & Bartlett Learning, LLC

NOT FOR SALE OR DISTRIBUTION 5′CH NOT FOR SALE OR DISTRIBUTION 2 N N NH2 O

3′ OH © Jones & Bartlett Learning, LLC © JonesO & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NaNOT+ – O FORP O SALE OR DISTRIBUTION O O NH U

3′ OH O

3′ end

© Jones & Bartlett Learning, LLC FIGURE 1.10 ©Segment Jones of & a polyribonucleotide.Bartlett Learning, (a) Extended LLC structure as a Tropp Molecular Biology of Genes NOTJones FOR and SALE Bartlett Publishers OR DISTRIBUTION sodium salt, (b)NOT stick FOR figure SALE structure, OR and DISTRIBUTION (c) an abbreviated structure. Elizabeth Morales Illustration Services Image10 09166-X_Ch01_Fig11.eps CHAPTER Date 02-01-07 1 INTRODUCTION TO MOLECULAR BIOLOGY 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORnucleotides SALE OR and DISTRIBUTION polypeptides are made of linear chainsNOT of FOR amino SALE acids OR DISTRIBUTION led to the sequence hypothesis, which proposes nucleotide sequences specify amino acid sequences. Drawing extended structures for DNA and RNA chains requires considerable time and space. It is more convenient to draw stick figure structures, which are© Jonesadequate & representationsBartlett Learning, for many LLC purposes © Jones & Bartlett Learning, LLC (Figure 1.9b and 1.10b).NOT FORThe fewSALE simple OR conventionsDISTRIBUTION for drawing NOT FOR SALE OR DISTRIBUTION stick figure representations for DNA and RNA are as follows: (1) a single line (shown as horizontal in Figures 1.9b and 1.10b) represents the pentose ring; (2) the letter A, G, C, U, or T, at one end of the line, represents© Jones the purine& Bartlett or pyrimidine Learning, attached LLC to C-1′ of the pentose© Jones & Bartlett Learning, LLC ring; NOT(3) the FOR letter SALE P, connected OR DISTRIBUTION by short diagonal lines to adjacent lines,NOT FOR SALE OR DISTRIBUTION represents the 5′→3′ phosphodiester bond; and (4) the symbol OH represents a hydroxyl group. An even simpler method for indicating nucleotide sequence is to write the letters corresponding to the bases (Figure 1.9c and 1.10c). © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

1.4 DNA: Hereditary Material Transformation experiments© Jones led & Bartlettto the discovery Learning, that LLCDNA is the © Jones & Bartlett Learning, LLC hereditary material.NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION The first step on the path leading to the discovery that genes are made of DNA was an observation made in 1928 by Fred Griffith, who was studying Streptococcus pneumoniae, a bacterial strain responsible for human© Jonespneumonia. & Bartlett This bacterium’s Learning, virulence LLC was known to depend© Jones & Bartlett Learning, LLC on aNOT surrounding FOR SALE polysaccharide OR DISTRIBUTION capsule that protects the bacteriumNOT FOR SALE OR DISTRIBUTION from the body’s defense systems. The capsule also causes the bacterium to produce smooth-edged colonies on an agar surface. Bacteria from smooth-edged colonies, called S bacteria, normally kill mice. Griffith © Jones &isolated Bartlett a S. Learning, pneumoniae LLC mutant that produced rough-edged© Jones & Bartlettcolo- Learning, LLC nies. Cells from rough-edged colonies, called R bacteria, proved to be NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION both non-encapsulated and nonlethal to mice. Additional experiments revealed that although either live R or heat-killed S bacteria are nonlethal to mice, a mixture of the two is lethal (FIGURE 1.11). Furthermore, when bacteria were isolated from a mouse that had died from such a mixed infection, the ©bacteria Jones were & Bartlett live S and Learning, R. Live R bacteria, LLC there- © Jones & Bartlett Learning, LLC fore, had somehow eitherNOT been FOR replaced SALE by OR or transformedDISTRIBUTION to S bacteria. NOT FOR SALE OR DISTRIBUTION Several years later investigators showed the mouse itself is not needed to mediate this transformation, because when a mixture containing live R bacteria and heat-killed S bacteria is incubated in culture medium,© Jones living & S Bartlett cells are Learning,produced. OneLLC possible explanation ©for Jones & Bartlett Learning, LLC this surprising phenomenon is that the R cells restore the viability of NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION the dead S cells. This hypothesis was eliminated by the observation that living S cells appear even when the heat-killed S bacteria in the mixture are replaced by an S cell extract, which had been centrifuged to remove both intact cells and the capsular polysaccharide. Based on © Jones &these Bartlett studies Learning, it appeared LLCthat the S cell extract contains© Jones a transforming & Bartlett Learning, LLC NOT FORprinciple SALE ORof unknown DISTRIBUTION chemical composition. NOT FOR SALE OR DISTRIBUTION

1.4 DNA: Hereditary Material 11 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

Living Living Heat-killed Living R cells plus S cells © Jones & BartlettR cells Learning, LLC S cells © Jonesheat-killed & Bartlett S cells Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Mouse contracts Mouse remains Mouse remains Mouse contracts pneumonia© Jones & Bartlett Learning,healthy LLC healthy © Jones & Bartlettpneumonia Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

S colonies isolated R colonies isolated No colonies isolated R and S colonies isolated from tissue of dead mouse from tissue from tissue from tissue of dead mouse © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC FIGURE 1.11 Griffith’s experiment demonstrating bacterial transformation. A mouse dies from pneumonia if injected with the virulent S (smooth) strainNOT of Streptococcus FOR SALE pneumoniae OR DISTRIBUTION. However, the mouse remains healthyNOT if injected FOR withSALE either OR the DISTRIBUTION nonvirulent R (rough) strain or the heat-killed S strain. R cells in the presence of heat-killed S cells are transformed into the virulent S strain, killing the mouse.

© Jones & Bartlett Learning, LLCThe next development ©occurred Jones in& Bartlett1944 when Learning, Oswald Avery,LLC NOT FOR SALE OR DISTRIBUTIONColin MacLeod, and MaclynNOT McCarty FOR determined SALE OR the DISTRIBUTION chemical nature of the transforming principle. They did so by purifying the transforming principle from S cells and adding it to live R bacterial cultures (FIGURE 1.12). After allowing the mixture to incubate for a period of time, they placed samples on an agar surface and incubated them until © Jones & Bartlett Learning, LLC colonies appeared.© Jones Some & Bartlettof the colonies Learning, (about LLC 1 in 104) that grew NOT FOR SALE OR DISTRIBUTION were S type. NOTTo show FOR this SALE was a ORpermanent DISTRIBUTION genetic change, Avery and

Tropp Molecular Biology of Genes coworkers dispersed many of the newly formed S colonies and placed Jones and Bartlett Publishers them on a second agar surface. The resulting colonies were again S Elizabeth Morales Illustration Services type. If an R colony arising from the original mixture was dispersed, Image 09166-X_Ch01_Fig12.eps Date 01-17-07 © Jones & Bartlettonly Learning,R bacteria grewLLC in subsequent generations.© Jones R colonies,& Bartlett hence, Learning, LLC NOT FOR SALEretained OR DISTRIBUTION the R character, whereas the transformedNOT FOR S colonies SALE bred OR true DISTRIBUTION as S cells. Chemical analysis showed the transforming principle was a deoxyribose-containing nucleic acid, and physical measurements showed it was a highly viscous substance having the properties of DNA. Because the transforming principle contained other kinds of © Jones & Bartlett Learning,molecules, LLC it was necessary© to Jones provide & evidence Bartlett that Learning, the transforma LLC - NOT FOR SALE OR DISTRIBUTIONtion was actually caused byNOT DNA FOR and not SALE an impurity. OR DISTRIBUTION This evidence was provided by the following three procedures: 1. Polysaccharides isolated from S cells did not transform R cells. 2. Incubation with trypsin or chymotrypsin, enzymes that © Jones & Bartlett Learning, LLC catalyze© Jones protein & hydrolysis,Bartlett Learning, or with ribonuclease LLC (RNase), NOT FOR SALE OR DISTRIBUTION an enzymeNOT FOR that SALEcatalyzes OR RNA DISTRIBUTION hydrolysis, did not affect

12 CHAPTER 1 INTRODUCTION TO MOLECULAR BIOLOGY 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

1. Disrupt cells 1. Precipitate with ethanol 2. Centrifuge 2. Redissolve in water © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Encapsulated S strain Cell-free Transforming extract principle © Jones & Bartlett Learning, LLC from S strain © Jones & Bartlett Learning, LLC

AdditionNOT FORof transforming SALE principleOR DISTRIBUTION to R strain NOT FOR SALE OR DISTRIBUTION

Culture containing R strain both S and R cells © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION FIGURE 1.12 Avery, MacLeod, and McCarty transformation experiment.

© Jonestransforming & Bartlett activity. Learning, The transforming LLC principle thus is nei© Jones- & Bartlett Learning, LLC NOTther FOR protein SALE nor ORRNA. DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 3. Incubation with deoxyribonuclease (DNase), an enzyme that catalyzes DNA hydrolysis, inactivated the transforming principle.

© Jones & BartlettIn drawing Learning, conclusions LLC from their experiments,© Avery, Jones MacLeod, & Bartlett Learning, LLC and McCarty did not explicitly state that DNA is the hereditary mate- NOT FORrial. SALE One ORreason DISTRIBUTION for their restraint may have been theNOT crude FOR analytical SALE OR DISTRIBUTION tools then available seemed to indicate that adenine, guanine, thymine, and cytosine were present in DNA in equimolar concentrations. Based on this and other evidence, many contemporary investigators mistak- enly thought DNA consisted© Jones of & a Bartlett repeating Learning, tetranucleotide LLC sequence © Jones & Bartlett Learning, LLC and so could not possiblyNOT exhibit FOR SALEthe variation OR DISTRIBUTION needed by the hereditary NOT FOR SALE OR DISTRIBUTION material. Instead, the prevailing theory was that genetic information was stored in proteins because protein composition and structure were known to vary among organisms. Investigators© Jones & whoBartlett supported Learning, the genes-as-protein LLC theory posed two© Jones & Bartlett Learning, LLC alternative explanations for the transformation results: (1) the trans- formingNOT principle FOR SALE might OR not DISTRIBUTIONbe DNA but rather one of the proteinsNOT FOR SALE OR DISTRIBUTION invariably contaminating the DNA sample and (2) DNA somehow affected capsule formation directly by acting in the metabolic pathway for biosynthesis of the polysaccharide and permanently altering Tropp Molecular Biology of Genes © Jones &this Bartlett pathway. Learning, The first LLCpoint should have already© beenJones discounted & Bartlett Learning, LLC Jones and Bartlett Publishers Elizabeth Morales Illustration Services NOT FORby SALE the original OR DISTRIBUTION work because the experiments showedNOT insensitivity FOR SALE to OR DISTRIBUTION Image 09166-X_Ch01_Fig13.eps Date 01-15-07 1.4 DNA: Hereditary Material 13 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION proteolytic enzymesNOT FOR and SALEsensitivity OR to DISTRIBUTION DNase. Because the DNase was not a pure enzyme, however, the possibility could not be eliminated conclusively. The transformation experiment was repeated 5 years later by Rollin Hotchkiss using a DNA sample with a protein content that was © Jones & Bartlettonly Learning, 0.02%, and LLC it was found that this extensive© Jones purification & Bartlett did Learning,not LLC NOT FOR SALEreduce OR DISTRIBUTION the transforming activity. This resultNOT supported FOR SALE the view OR ofDISTRIBUTION Avery, MacLeod, and McCarty but still did not prove it. The second alternative, however, was clearly eliminated—also by Hotchkiss—with an experiment in which he transformed a penicillin-sensitive bacterial © Jones & Bartlett Learning,strain LLC to penicillin resistance.© JonesBecause & penicillin Bartlett resistance Learning, is totallyLLC NOT FOR SALE OR DISTRIBUTIONdistinct from the rough-smoothNOT character FOR SALE of the ORbacterial DISTRIBUTION capsule, this experiment showed the transforming ability of DNA was not limited to capsule synthesis. Interestingly enough, most biologists still remained unconvinced that DNA was the genetic material. It was not until Erwin Chargaff showed in 1950 that a wide variety of chemical structures © Jones & Bartlett Learning, LLC in DNA were© possible—thus Jones & Bartlett allowing Learning, biological LLC specificity—that this NOT FOR SALE OR DISTRIBUTION idea was accepted.NOT FOR SALE OR DISTRIBUTION

Chemical experiments also supported the hypothesis that DNA is © Jones & Bartlettthe hereditaryLearning, material. LLC © Jones & Bartlett Learning, LLC NOT FOR SALEThe OR hypothesis DISTRIBUTION of a tetranucleotide structureNOT for FORDNA SALEarose for OR two DISTRIBUTION reasons. First, in the chemical analysis of DNA, the technique used to separate the bases before identification did not resolve them very well, so the quantitative analysis was poor. Second, the DNA analyzed was usually isolated from animals, plants, and yeast in which the four bases © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC are present in nearly equimolar concentration or from bacterial species NOT FOR SALE OR DISTRIBUTIONsuch as Escherichia coli that NOTalso happen FOR SALEto have ORnearly DISTRIBUTION equimolar base concentrations. Using the DNA from a wide variety of organisms, Char- gaff applied new separation and analytical techniques and showed that the molar content of bases (generally called the base composition) could © Jones & Bartlett Learning, LLC vary widely. ©The Jones base composition & Bartlett of Learning, DNA from LLC a particular organism NOT FOR SALE OR DISTRIBUTION is usually expressedNOT FOR as a fractionSALE OR of all DISTRIBUTION bases that are G · C pairs. This fraction, called the G + C content, can be expressed as follows:

G + C content = ([G] + [C])/[all bases]

© Jones & Bartlettwhere Learning, the square LLC brackets ([ ]) denote molar© Jones concentrations. & Bartlett Base Learning, LLC NOT FOR SALEcompositions OR DISTRIBUTION of DNAs from many differentNOT organisms FOR SALE have OR been DISTRIBUTION determined. Generally speaking, the value of the G + C content is near 0.50 for the higher organisms, ranging from 0.49 to 0.51 for primates. For lower organisms the value of the G + C content var- © Jones & Bartlett Learning,ies LLC widely from one genus ©to Jonesanother. & For Bartlett example, Learning, for bacteria LLC the extremes are 0.27 for the genus Clostridium and 0.76 for the genus NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Sarcina; E. coli DNA has the value 0.50. Thus, it was demonstrated that DNA could have variable composition, a primary requirement for the hereditary material. Chargaff’s studies also revealed one other remarkable fact about © Jones & Bartlett Learning, LLC DNA base composition.© Jones & InBartlett each of Learning, the DNA samples LLC Chargaff stud- NOT FOR SALE OR DISTRIBUTION ied, he foundNOT that FOR [A] = SALE [T] and OR [G] DISTRIBUTION = [C]. Chargaff’s findings can

14 CHAPTER 1 INTRODUCTION TO MOLECULAR BIOLOGY 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORbe SALE summarized OR DISTRIBUTION by two rules, known as Chargaff’s NOTrules, FORwhich SALE state: OR DISTRIBUTION (1) double stranded DNA has equimolar adenine and thymine concentrations as well as equimolar guanine and cytosine concentrations; and (2) DNA composition varies from one genus to another. Although the significance of Chargaff’s rules, was not immediately apparent, they would later help© toJones confirm & Bartlettthe structure Learning, of the DNA LLC molecule. © Jones & Bartlett Learning, LLC Upon publicationNOT of Chargaff’s FOR SALE results, OR the DISTRIBUTION tetranucleotide hypoth- NOT FOR SALE OR DISTRIBUTION esis quietly died and the DNA–gene idea began to catch on. Shortly afterward, workers in several laboratories found for a wide variety of organisms that somatic cells have twice the DNA content of germ cells,© a Jonescharacteristic & Bartlett to be Learning, expected of LLC the genetic material, given© Jones & Bartlett Learning, LLC the tenetsNOT ofFOR classical SALE chromosome OR DISTRIBUTION genetics. Although it could applyNOT FOR SALE OR DISTRIBUTION just as well to any component of chromosomes, once this result was revealed objections to the work of Avery, MacLeod, and McCarty were no longer heard, and the hereditary nature of DNA rapidly became the accepted idea. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

1.5 Watson-Crick Model Rosalind Franklin and© Jones Maurice & WilkinsBartlett obtained Learning, x-ray LLC diffraction © Jones & Bartlett Learning, LLC patterns of extendedNOT DNA FOR fibers. SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION At the same time chemists were attempting to learn something about the composition of DNA, crystallographers were trying to obtain a three-dimensional image of the molecule. Rosalind Franklin and Maurice© Jones Wilkins & Bartlettobtained Learning,some excellent LLC x-ray diffraction patterns© Jones & Bartlett A-formLearning, DNA LLC of extendedNOT FOR DNA SALE fibers OR in the DISTRIBUTION early 1950s (FIGURE 1.13). One mightNOT FOR SALE OR DISTRIBUTION predict that all x-ray diffraction patterns would look alike, but this was not the case. DNA structure and therefore x-ray diffraction patterns depend on several variables. One of the most important of these is © Jones &the Bartlett relative humidityLearning, of theLLC chamber in which DNA© fibers Jones are & placed. Bartlett Learning, LLC Two types of DNA structure are of particular interest. B-DNA is NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION stable at a relative humidity of about 92%, whereas A-DNA appears as the relative humidity falls to about 75%. Crystallographers did not know whether A-DNA or B-DNA was present in the living cell. Partly for this reason, Wilkins turned his attention toward taking x-ray diffraction pictures of© DNAJones in sperm& Bartlett cells. FranklinLearning, focused LLC her atten- © Jones & Bartlett Learning, LLC tion on x-ray diffractionNOT patterns FOR SALE of A-DNA OR becauseDISTRIBUTION they appeared to NOT FOR SALE OR DISTRIBUTION provide more detail. She believed that careful analyses of the detailed patterns would eventually lead to the solution of DNA’s structure.

James© JonesWatson & and Bartlett Francis Learning, Crick proposed LLC that DNA is a © Jones & Bartlett Learning, LLC B-form DNA double-strandedNOT FOR SALE helix. OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION The American biologist, James D. Watson, and the English crystal- FIGURE 1.13 X-ray diffraction patterns of the lographer, Francis H. C. Crick, working together in England, took A and B forms of the sodium salt of DNA. (Reproduced from Franklin, R. E., and Gosling, a different approach to determining DNA’s structure. They tried to R. G. 1953. Acta Crystallographica 6:673–677. © Jones &obtain Bartlett as much Learning, information LLC as they could from the© x-rayJones diffraction & Bartlett Photos Learning, courtesy LLC of International Union of NOT FORpatterns SALE andOR thenDISTRIBUTION to build a model consistent withNOT this information.FOR SALE ORCrystallography.) DISTRIBUTION

1.5 Watson-Crick Model 15

4TH PAGES Tropp Molecular Biology of Genes Jones and Bartlett Publishers Image 09166-X_Ch01_Fig16.eps Date 06-21-07 © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION The termNOT “model” FOR has SALE a special OR meaningDISTRIBUTION to scientists. A model is a hypothesis or tentative explanation of the way a system works, usually including the components, interactions, and sequences of events. A successful model suggests additional experiments and allows investigators to make predictions that can be tested in the laboratory. If © Jones & Bartlettpredictions Learning, do not LLC agree with experimental© results,Jones the& Bartlett model must Learning, LLC NOT FOR SALEbe OR considered DISTRIBUTION incorrect in its current formNOT and FORmodified. SALE A ORmodel DISTRIBUTION cannot be proved to be correct merely by showing it makes a correct prediction. If it makes many correct predictions, however, it is prob- ably nearly, if not completely, correct. © Jones & Bartlett Learning, LLCWatson and Crick focused© Jones their &attention Bartlett on Learning, Franklin’s LLC x-ray NOT FOR SALE OR DISTRIBUTIONdiffraction patterns of B-DNA.NOT This FOR pattern SALE indicated OR DISTRIBUTION that B-DNA has a helical structure, a diameter of approximately 2.0 nm, and a repeat distance of 0.34 nm. Their model would have to account for these structural features. Watson and Crick still had to work out the number of DNA chains in a DNA molecule, the location of the bases, © Jones & Bartlett Learning, LLC and the position© Jones of the phosphate& Bartlett and Learning, deoxyribose LLC groups. The density NOT FOR SALE OR DISTRIBUTION of DNA seemedNOT to FOR be consistent SALE OR with DISTRIBUTION one, two, or three chains per molecule. Watson and Crick tried to build a two-chain model with hydrogen bonds (weak electrostatic attractions; see Chapter 2) holding the bases together. They were unsuccessful until their colleague, Jerry © Jones & BartlettDonohue, Learning, suggested LLC they use the keto tautomeric© Jones forms & Bartlett of T and Learning, G LLC NOT FOR SALEin OR their DISTRIBUTION models. At first, Watson tried to NOTlink two FOR purines SALE together OR DISTRIBUTION and two pyrimidines together in what he called a “like-with-like” model. A dramatic turning point occurred in 1953 when Watson real- ized adenine forms hydrogen bonds with thymine and guanine forms hydrogen bonds with cytosine. Watson describes this turning point © Jones & Bartlett Learning,in LLC his book, The Double Helix© Jones (pages & 194–195): Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION When I got to our still empty office the following morning, I quickly cleared away the paper from my desk top so that I would have a large, flat surface on which to form pairs of bases held together → by hydrogen bonds. Though I initially went back to my like-with-

Jerry [Donohue] came in I looked up, saw that it was not Francis NOT FOR SALE OR DISTRIBUTION→ [Crick], andNOT began FOR shifting SALE the OR bases DISTRIBUTION in and out of various other pairing possibilities. Suddenly I became aware that an adenine- thymine pair held together by two hydrogen bonds was identical in shape to a guanine-cytosine base pair held together by at least © Jones & Bartletttwo Learning, hydrogen LLC bonds. All the hydrogen bonds© Jones seemed & Bartlett to form Learning, LLC naturally; no fudging was required to make the two types of base NOT FOR SALE ORpairs DISTRIBUTION identical in shape. NOT FOR SALE OR DISTRIBUTION With the realization that adenine-thymine and cytosine-guanine base pairs have the same width, Watson and Crick were quickly able to construct a double helix model of DNA that fit Franklin’s x-ray © Jones & Bartlett Learning,diffraction LLC data (FIGURE 1.14©). Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONThe key features of theNOT Watson-Crick FOR SALE Model OR forDISTRIBUTION B-DNA are as follows: 1. Two polydeoxyribonucleotide strands twist about each other FIGURE 1.14 DNA structure based on the 1953 paper by Watson and Crick in Nature to form a double helix. © Jonesshowing & theirBartlett double-helix Learning, model LLC for DNA for 2. Phosphate© Jones and &deoxyribose Bartlett Learning, groups form LLC a backbone on the NOTthe FOR first time.SALE OR DISTRIBUTION outsideNOT of FORthe helix. SALE OR DISTRIBUTION

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE 3. Purine OR DISTRIBUTION and pyrimidine base pairs stack insideNOT the FOR helix SALE and OR DISTRIBUTION form planes perpendicular to the helix axis and the deoxyribose groups. 4. The helix diameter is 2.0 nm (or 20 Å). 5. Adjacent base pairs are separated by an average distance of 0.34 nm (or ©3.4 Jones Å) along & Bartlettthe helix axis.Learning, The structure LLC repeats © Jones & Bartlett Learning, LLC itself after aboutNOT 10FOR base SALE pairs, OR or aboutDISTRIBUTION once every 3.4 nm NOT FOR SALE OR DISTRIBUTION (or 34 Å) along the helix axis. 6. Adenine always pairs with thymine and guanine with cytosine. The original model showed two hydrogen bonds stabilizing © Joneseach kind & ofBartlett base pair. Learning, Although LLC this was an accurate descrip© -Jones & Bartlett Learning, LLC NOTtion FOR for A-T SALE base OR pairs, DISTRIBUTION later work showed that G-C base pairsNOT FOR SALE OR DISTRIBUTION are stabilized by three hydrogen bonds (FIGURE 1.15). (These base pairing relationships explained Chargaff’s observation that the molar ratios of adenine to thymine and guanine to cytosine are one.) © Jones & Bartlett 7. The twoLearning, strands areLLC antiparallel, which means© Jones the strands & Bartlett run Learning, LLC NOT FOR SALEin OR opposite DISTRIBUTION directions. That is, one strand NOTruns 3FOR′→5′ SALEin one OR DISTRIBUTION direction, whereas the other strand runs 5′→3′ in the same direction. Because the strands are antiparallel, a convention is needed for stating the sequence of bases of a single chain. The convention is© toJones write a& sequence Bartlett with Learning, the 5′ terminus LLC at the © Jones & Bartlett Learning, LLC left; for example,NOT FORATC denotesSALE OR the DISTRIBUTIONtrinucleotide 5′-ATC-3′. NOT FOR SALE OR DISTRIBUTION This is also often written as pApTpC, again using the conven- tions that the left side of each base is the 5′-terminus of the nucleotide and that a phosphodiester group is represented by a “p” between two capital letters. ©8. JonesA major & groove Bartlett and Learning,a minor groove LLC wind about the cylindrical© Jones & Bartlett Learning, LLC NOTouter FOR helical SALE face. OR The DISTRIBUTION two grooves are of about equal depth,NOT FOR SALE OR DISTRIBUTION but the major groove is much wider than the minor groove. The Watson-Crick model indicates that when the nucleotide sequence of one strand is known, the sequence of the complementary © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION H

NH N H3C O Adenine Thymine N H N N © Jones & Bartlett Learning,O LLC © Jones & Bartlett Learning, LLC N N O NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION O Deoxyribose Deoxyribose H © Jones & BartlettN Learning,H O LLCN © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION Guanine NOT FOR SALE OR DISTRIBUTION Cytosine N H N A N O N O N O NH Deoxyribose © Jones & BartlettDeoxyribose Learning, LLC H © Jones & Bartlett Learning, LLC NOT FORFIGURE SALE 1. OR15 Base DISTRIBUTION pairs in DNA. NOT FOR SALE OR DISTRIBUTION

1.5 Watson-Crick Model 17 4TH PAGES

Tropp Molecular Biology of Genes Jones and Bartlett Publishers Elizabeth Morales Illustration Services Image 09166-X_Ch01_Fig18.eps Date 01-15-07 © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION strand can beNOT predicted, FOR SALEproviding OR the DISTRIBUTION theoretical framework needed to understand the fidelity of gene replication. Each strand serves as a mold or template for the synthesis of the complementary strand (FIGURE 1.16). Watson and Crick ended their short paper announcing the double helix model with the following sentence that must be one © Jones & Bartlettof the Learning, greatest understatements LLC in the scientific© Jones literature: & Bartlett “It has Learning,not LLC NOT FOR SALEescaped OR DISTRIBUTION our notice that the specific pairing weNOT have FOR postulated SALE immedi OR DISTRIBUTION- ately suggests a possible copying mechanism for the genetic material.” The double helix showed how establishing a chemical structure can be used to understand biological function and to make predictions © Jones & Bartlett Learning,that LLC guide new research. © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONStructure–function relationshipsNOT FOR remain SALE a central OR DISTRIBUTIONtheme of molecular biology. Knowledge of structure helps us to understand function

TA © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,CG LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE ORAT DISTRIBUTION

CG CG Parent duplex © Jones & Bartlett Learning, LLC GC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION T ANOT FOR SALE OR DISTRIBUTION

CG T A CG © Jones & Bartlett Learning, LLC © JonesTA & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION TA A T A T C G © Jones & Bartlett Learning, LLC © Jones & BartlettC G Learning,CG LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTIONGC G C T A T Daughter A duplex CG C G T A T A © Jones & Bartlett Learning, LLC C G © JonesCG & Bartlett Learning, LLC T A NOT FOR SALE OR DISTRIBUTION NOTTA FOR SALE OR DISTRIBUTION

T A TA Template A T strands AT © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Replica strands

FIGURE 1.16 Replication of DNA. Replication of a DNA duplex as originally envisioned by Watson and Crick. As the parental strands separate, each © Jones & Bartlett Learning, LLC parental strand© servesJones as &a templateBartlett for Learning, the formation LLC of a new daughter NOT FOR SALE OR DISTRIBUTION strand by meansNOT of A-TFOR and SALE G-C base OR pairing. DISTRIBUTION

18 CHAPTER 1 INTRODUCTION TO MOLECULAR BIOLOGY 4TH PAGES

Tropp Molecular Biology of Genes Jones and Bartlett Publishers Elizabeth Morales Illustration Services Image 09166-X_Ch01_Fig19.eps Date 01-17-07 © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORand SALE leads OR us to DISTRIBUTION new insights. Biological structures areNOT sometimes FOR SALE quite OR DISTRIBUTION complex and difficult to study. It is, however, worth the effort to study the structures because the reward for doing so is so great. The Watson-Crick model also serves as a powerful example of the fundamental principles that help to define molecular biology as a discipline: (1) the same physical© andJones chemical & Bartlett laws apply Learning, to living LLC systems and © Jones & Bartlett Learning, LLC inanimate objects, (2)NOT the FORsame biologicalSALE OR principles DISTRIBUTION tend to apply to NOT FOR SALE OR DISTRIBUTION all organisms, and (3) biological structure and function are intimately related. The Watson-Crick model provided an excellent starting point for the challenging job of elucidating the chemical basis of heredity. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

1.6 Central Dogma © Jones &The Bartlett central Learning,dogma provides LLC the theoretical framework© Jones for & Bartlett Learning, LLC NOT FORmolecular SALE OR biology. DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Much of the research in molecular biology in the late 1950s was directed toward discovering the mechanisms by which nucleotide sequences specify amino acid sequences. In a talk given at a 1957 sym- posium, Crick suggested© Jones a model & Bartlett for information Learning, flow LLC that provides © Jones & Bartlett Learning, LLC a theoretical frameworkNOT for FOR molecular SALE biology. OR DISTRIBUTION The following excerpt NOT FOR SALE OR DISTRIBUTION from Crick’s presentation (“On Protein Synthesis.” The Symposia of the Society for Experimental Biology 12, (1958):138–163) states the theory known as the central dogma in a clear and concise fashion: In© moreJones detail, & Bartlett the transfer Learning, of information LLC from nucleic acid to © Jones & Bartlett Learning, LLC nucleicNOT FORacid, orSALE from nucleicOR DISTRIBUTION acid to protein may be possible, but NOT FOR SALE OR DISTRIBUTION DNA transfer from protein to protein, or from protein to nucleic acid is impossible. Information means here the precise determination of sequence, either of bases in nucleic acid or of amino acid residues Replication in protein. Transcription © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Crick thus was proposing that genetic information flows from DNA mRNA NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION to DNA (DNA replication), from DNA to RNA (transcription), and FIGURE 1.17 from RNA to polypeptide (translation) ( ). Translation By the mid-1960s molecular biologists had obtained considerable experimental support for the central dogma. In particular, they had Ribosome discovered enzymes© that Jones catalyze & Bartlett replication Learning, and transcription LLC and © Jones & Bartlett Learning, LLC elucidated the pathwayNOT for FOR translating SALE nucleotide OR DISTRIBUTION sequences to amino NOT FOR SALE OR DISTRIBUTION acid sequences. With some variations in detail, all organisms use

the same basic mechanism to translate information from nucleotide Polypeptide sequences to amino acid sequences. Three major components of the translation© Jones machinery— & Bartlettmessenger Learning, RNA LLC (mRNA), ribosomes, and© Jones & Bartlett Learning, LLC transfer RNA (tRNA)—play essential roles in this information trans- NOT FOR SALE OR DISTRIBUTION NOT FIGUREFOR SALE 1.17 The OR “central DISTRIBUTION dogma.” The fer (FIGURE 1.18). Each gene can serve as a template for the synthesis central dogma as originally proposed by of specific mRNA molecules. mRNA molecules program ribosomes Francis Crick postulated information flow from (protein synthetic factories) to form specific polypeptides. tRNA DNA to RNA to protein. The ribosome is an essential part of the translation machinery. molecules carry activated amino acids to programmed ribosomes, Later studies demonstrated that information © Jones &where Bartlett under Learning, the direction LLC of the mRNA the amino© acids Jones join &to Bartlettform can Learning, also flow from LLC RNA to RNA and from RNA NOT FORa SALEpolypeptide OR DISTRIBUTION chain. NOT FOR SALE ORto DNA DISTRIBUTION (reverse transcription).

mRNA Transcription Growing polypeptide chain © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION tRNANOT FOR SALE OR DISTRIBUTION

Incoming tRNA with amino acid Outgoing © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC tRNA NOT FOR SALE OR DISTRIBUTION TranslationNOT FOR SALE OR DISTRIBUTION

mRNA

Ribosome © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC FIGURE 1.18 Simplified schematic diagram of the protein synthetic machinery in an animal, plant, or yeast cell. Transfer RNA (tRNA) carries the next aminoNOT acidFOR to SALEbe attached OR to DISTRIBUTION the growing polypeptide chain to the ribosome,NOT FOR which SALE recognizes OR DISTRIBUTION a match between a three nucleotide sequence in the mRNA (the codon) and a complementary sequence in the tRNA (the anticodon) and transfers the growing polypeptide chain to the incoming amino acid while it is still attached to the tRNA. (Adapted from Secko, D. 2007. The Science Creative Quarterly 2. http://www.scq.ubc.ca/a-monks-flourishing-garden-the- basics-of-molecular-biology-explained/, Figure 4.) © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION The 12 years of extraordinary scientific progress that followed the discovery of DNA’s structure were capped by a series of brilliant investigations by Marshal W. Nirenberg and others that culminated in deciphering the genetic code. By 1965 investigators could predict © Jones & Bartlett Learning, LLC a polypeptide© chain’sJones amino& Bartlett acid sequenceLearning, from LLC a DNA or mRNA NOT FOR SALE OR DISTRIBUTION molecule’s nucleotideNOT FOR sequence. SALE OR Remarkably, DISTRIBUTION the genetic code was found to be nearly universal. Each particular sequence of three adjacent nucleotides or codon specifies the same amino acid in bacteria, plants, and animals. Could one ask for more convincing support for © Jones & Bartlettthe uniformity Learning, of LLC life processes? © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Introduction to Recombinant DNA © Jones & Bartlett Learning,1.7 LLC Technology © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Recombinant DNA technology allows us to study complex biological systems. The second major wave in molecular biology started in the late © Jones & Bartlett Learning, LLC 1970s with© the Jones development & Bartlett of Learning,recombinant LLC DNA technology

NOTTropp FOR Molecular SALE Biology OR of DISTRIBUTION Genes (also knownNOT as genetic FOR engineeringSALE OR ).DISTRIBUTION Thanks to recombinant DNA Jones and Bartlett Publishers Elizabeth20 Morales Illustration ServicesCHAPTER 1 INTRODUCTION TO MOLECULAR BIOLOGY Image 09166-X_Ch01_Fig21.eps Date 01-15-07 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORtechnology SALE OR genes DISTRIBUTION can be manipulated in the laboratoryNOT FORjust like SALE any OR DISTRIBUTION other organic molecule. They can be synthesized or modified as desired and their nucleotide sequence determined. Sequence information can save molecular biologists months and perhaps even years of work. For example, when a new gene is discovered that causes a specific disease in© humans, Jones &molecular Bartlett biologistsLearning, may LLC be able to © Jones & Bartlett Learning, LLC obtain valuable cluesNOT to theFOR new SALE gene’s OR function DISTRIBUTION by comparing its NOT FOR SALE OR DISTRIBUTION nucleotide sequence with sequences of all other known genes. If similarities are found with a gene of known function from some other organism, then it is likely the new gene will have a similar function.© Jones Alternatively, & Bartlett a segment Learning, of the LLC nucleotide sequence of ©the Jones & Bartlett Learning, LLC new NOTgene may FOR predict SALE an OR amino DISTRIBUTION acid sequence that is known to haveNOT FOR SALE OR DISTRIBUTION specific binding or catalytic functions. Development of recombinant DNA technology has allowed the incredible progress in solving problems that once seemed intracta- ble. Recombinant DNA technology has helped investigators to study © Jones &cell Bartlett division, Learning, cell differentiation, LLC transformation ©of Jonesnormal &cells Bartlett to Learning, LLC NOT FORcancer SALE cells, OR programmedDISTRIBUTION cell death (apoptosis), NOTantibody FOR produc SALE- OR DISTRIBUTION tion, hormone action, and a variety of other fundamental biological processes. One of the most exciting applications of recombinant DNA technology is in medicine.© Jones Until & Bartlett relatively Learning, recently many LLC diseases © Jones & Bartlett Learning, LLC could only be studiedNOT in humans FOR SALE because OR no DISTRIBUTION animal models existed. NOT FOR SALE OR DISTRIBUTION Very little progress was made in studying these diseases because of obvious ethical constraints associated with studying human diseases. Once investigators learned how to modify and transfer genes, they were able to create animal models for human diseases, thus facilitat- ing study© Jones of the & diseases. Bartlett Recombinant Learning, LLCDNA technology also has ©led Jones & Bartlett Learning, LLC to theNOT production FOR SALE of new OR drugs DISTRIBUTION to treat diabetes, anemia, cardiovasNOT- FOR SALE OR DISTRIBUTION cular disease, and cancer as well as to the development of diagnostic tools to detect a wide variety of diseases. The list of practical medical applications grows longer with each passing day. © Jones & BartlettAlthough Learning, recombinant LLC DNA technology promises© Jones to change & Bartlett our Learning, LLC NOT FORlives SALE for ORthe better,DISTRIBUTION it also forces us to consider NOTimportant FOR social,SALE OR DISTRIBUTION political, ethical, and legal issues. For example, how do we protect the interests of an individual when DNA analysis reveals the individual has alleles likely to cause a serious physical or mental disease in the future, especially if there is no cure or treatment? What impact will the knowledge have ©on Jones affected & individuals Bartlett Learning,and their families? LLC Should © Jones & Bartlett Learning, LLC insurance companiesNOT or potential FOR SALE employers OR haveDISTRIBUTION access to the genetic NOT FOR SALE OR DISTRIBUTION information? If not, how do we limit access to information and how do we enforce the limitation? Rapid progress in recombinant DNA technology also raises troubling ethical issues. Germ line therapy allows new ©genes Jones to be & introducedBartlett Learning, into fertilized LLC eggs and thereby alter ©the Jones & Bartlett Learning, LLC geneticNOT characteristics FOR SALE of OR future DISTRIBUTION generations. This technique has beenNOT FOR SALE OR DISTRIBUTION used to introduce desired traits into plants and animals. An argument can be made for using germ line therapy to correct human genetic diseases, such as Tay-Sachs disease, cystic fibrosis, or Huntington disease, so affected individuals and their families can © Jones &be Bartlettspared the Learning, devastating LLC consequences of these diseases.© Jones However, & Bartlett Learning, LLC NOT FORwe SALE must ORbe very DISTRIBUTION careful about application of germNOT line FOR therapy SALE to OR DISTRIBUTION

1.7 Introduction to Recombinant DNA Technology 21 4TH PAGES © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION.

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION humans becauseNOT the FOR technique SALE has OR the DISTRIBUTION power to do great harm. Who will decide which genetic characteristics are desirable and which are undesirable? Should the technique be used to change physical appear- ance, intelligence, or personality traits? Should anyone be permitted to make such decisions? © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALEA OR great DISTRIBUTION deal of molecular biology informationNOT is FOR available SALE OR DISTRIBUTION on the Internet. Recombinant DNA technology has generated so much information it would be nearly impossible to share all of it in a timely fashion with © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC the entire molecular biology community by conventional means such NOT FOR SALE OR DISTRIBUTIONas publishing in professionalNOT journals FOR or SALE writing OR books. DISTRIBUTION Fortunately, there is an alternate method for sharing large quantities of rapidly accumulating information that is both quick and efficient. A world- wide network of communication networks, the Internet, allows us © Jones & Bartlett Learning, LLC to gain almost© Jones instant &access Bartlett to the Learning, information. LLC The Internet also NOT FOR SALE OR DISTRIBUTION provides manyNOT helpful FOR tutorials SALE ORand DISTRIBUTIONinstructive animations. This text will include references to helpful Internet sites. Because the addresses for these Web sites tend to change over time, however, this text will refer the reader to a primary site maintained by the publisher. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALEQuestions OR DISTRIBUTION and Problems NOT FOR SALE OR DISTRIBUTION

1. Briefly define, describe, or illustrate each of the following terms. a. Allele m. Transformation b. Phenotype o. Transforming principle © Jones & Bartlett Learning, LLC c. Genotype © Jones &p. Chargaff’sBartlett rulesLearning, LLC NOT FOR SALE OR DISTRIBUTION d. Dominant gene NOT FORq. SALE B-DNA OR DISTRIBUTION e. Recessive gene r. Model f. Purine s. Major groove g. Pyrimidine t. Minor groove h. Nucleoside u. Replication © Jones & Bartlett Learning, LLC i. N-glycosylic© Jones bond & Bartlett Learning,v. Transcription LLC j. Nucleotide w. Translation NOT FOR SALE OR DISTRIBUTION k. PhosphomonoesterNOT FOR SALE OR DISTRIBUTIONx. Codon l. Phosphodiester bond 2. Draw the four bases commonly found in DNA. Which of these bases is a purine and which is a pyrimidine? 3. Draw the base that is commonly present in RNA but not in DNA. Is this base a © Jones & Bartlettpurine Learning, or a pyrimidine? LLC © Jones & Bartlett Learning, LLC NOT FOR SALE 4.OR What DISTRIBUTION are the chemical similarities and differences betweenNOT FOR ribose andSALE deoxyribose? OR DISTRIBUTION 5. Each purine and pyrimidine base in nucleic acids has distinct chemical features. a. Which purine has a single amine group and no oxygen atoms? b. Which pyrimidine has a single amine group and one oxygen atom? © Jones & Bartlett Learning, LLC c. What is the major difference© betweenJones the & structures Bartlett of Learning,uracil and thymine? LLC d. Which pyrimidine has two oxygen atoms attached to the ring structure? NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 6. Although Avery, MacLeod, and McCarty did not explicitly state that DNA is the hereditary material, their experiment certainly seemed to show this was the case. a. Describe their experiment. b. Why were their experiments showing that the transforming principle is resistant to RNase and proteases but sensitive to DNase important? © Jones & Bartlett Learning, LLC c. Why were© contemporary Jones & Bartlett investigators Learning, slow to accept LLC the idea that DNA is the NOT FOR SALE OR DISTRIBUTION hereditaryNOT material? FOR SALE OR DISTRIBUTION

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE d. Why OR did Chargaff’s DISTRIBUTION studies of DNA base composition helpNOT to convinceFOR SALE inves- OR DISTRIBUTION tigators that DNA is the hereditary material? e. How does the Watson-Crick model explain Chargaff’s rules? 7. DNA and RNA are linear nucleotide chains. a. Draw the extended structure for a segment of a polydeoxyribonucleotide with the abbreviated structure© Jones pdTpdApdC. & Bartlett Indicate Learning, the 5′- and 3LLC′-ends. © Jones & Bartlett Learning, LLC b. Draw the stick figure structure for a segment of a polydeoxyribonucleotide with the abbreviatedNOT structure FOR pdTpdApdC. SALE OR Indicate DISTRIBUTION the 5′- and 3′-ends. NOT FOR SALE OR DISTRIBUTION c. Draw the extended structure for a segment of a polyribonucleotide with the abbreviated structure pUpApC. Indicate the 5′- and 3′-ends. d. Draw the stick figure structure for a segment of a polyribonucleotide with the abbreviated structure pUpApC. Indicate the 5′- and 3′-ends. e. ©Identify Jones two important& Bartlett chemical Learning, differences LLC between a polydeoxyribonucleotide© Jones & Bartlett Learning, LLC NOTand a polyribonucleotideFOR SALE OR strand. DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 8. What are the major structural features of the Watson-Crick model for DNA? 9. How does the Watson-Crick model help us to understand DNA function? 10. What functions do ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA) play in the cell? © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Suggested Reading

General Choudri, S. 2003. The path© Jones from nuclein & Bartlett to human Learning,genome: a brief LLC history of DNA © Jones & Bartlett Learning, LLC with a note on humanNOT genome FOR sequencing SALE and OR its DISTRIBUTION impact on future research in NOT FOR SALE OR DISTRIBUTION biology. Bull Sci Technol Soc 23:360–367. Crick, F. 1974. The double helix: a personal view. Nature 248:766–769. Crick, F. 1988. What Mad Pursuit: A Personal View of Scientific Discovery. New York: Basic Books. Judson,© JonesH. F. 1996. & BartlettThe Eighth Learning, Day of Creation: LLC Makers of the Revolution© in Jones & Bartlett Learning, LLC Biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Maddox,NOT B. 2002.FOR Rosalind SALE Franklin:OR DISTRIBUTION The Dark Lady of DNA. New York: HarperNOT FOR SALE OR DISTRIBUTION Collins. McCarty, M. 1985. The Transforming Principle: Discovering That Genes Are Made of DNA. New York: W. W. Norton. Olby, R. C. 1994. The Path to the Double Helix: The Discovery of DNA. New © Jones & York:Bartlett Dover. Learning, LLC © Jones & Bartlett Learning, LLC Pukkila, P. J. 2001. Molecular biology: the central dogma. Encyclopedia of Life NOT FOR SALESciences .OR pp. 1–5.DISTRIBUTION London, UK: Nature. NOT FOR SALE OR DISTRIBUTION Sayre, A. 1978. Rosalind Franklin and DNA. New York: W. W. Norton. Stent, G. 1972. Prematurity and uniqueness in scientific discovery. Sci Am 227:84–93. Summers, W. C. 2002. History of molecular biology. Encyclopedia of Life Sciences. pp. 1–8. London, UK: Nature. Watson, J. D. 1968. The© Double Jones Helix: & ABartlett Personal AccountLearning, of the LLCDiscovery of the © Jones & Bartlett Learning, LLC Structure of DNA. NewNOT York: FOR Antheneum SALE Books. OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Wilkins, M. 2003. The Third Man of the Double Helix: The Autobiography of Maurice Wilkins. Oxford, UK: Oxford University Press.

Classic© Jones Papers & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Avery,NOT O. T., FOR Macleod, SALE C. M., OR and McCarty,DISTRIBUTION M. 1944. Studies on the chemical natureNOT FOR SALE OR DISTRIBUTION of the substance inducing transformation of pneumococcal types. Induction of transformation by a deoxyribonucleic acid fraction isolated from Pneumococcus type III. J Exp Med 79:137–156. Beadle, G. W., and Tatum, E., 1941. Genetic control of biochemical reactions in Neurospora. Proc Natl Acad Sci USA 27:499–506. © Jones &Watson, Bartlett J. D., andLearning, Crick F. H. LLC 1953. Molecular structure of nucleic© Jones acids; a & structure Bartlett Learning, LLC NOT FOR SALEfor deoxyribose OR DISTRIBUTION nucleic acid. Nature 171:737–738. NOT FOR SALE OR DISTRIBUTION