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When mixtures of nucleotides are present,le wavelength at 260 nm (dashed vertical lines) issed for measurements." v NAo12,000"•3^10,000Jj8,00016,000loJed4,0002,000/////-//////\AMP\ ^<. \\ \+s£So\\\\\V7\\CMP6,000034,000'o/ ///\\\11\\2,000230 240 250 260 270 280Wavelength (nm)dTMPI10,00003\1UMPo\\i12,0008,000\ \\7/iMolar absorptioncoefficient of nucleotides, e26o(M~1cm~1)14,000 -)sor]GMP•±,V\JVJ>\1I\i1230 240 250 260 270 280Wavelength (nm)AMPGMPCMPUMPdTMP15,40011,7007,5009,9009,200Chapter 12 Nucleotides and Nucleic Acids331Figure 12-11 Hydrogenbonding patterns in thebase pairs defined byWatson and Crick.H-CAdeninec-rThyminec-rH~CCytosineGuaninec-rtween bases permit a complementary association of two and occasionally three strands of nucleic acid.
The most important hydrogen-bonding patterns are those defined by James Watson and Francis Crick in1953, in which A bonds specifically to T (or U) and G bonds to C (Fig.12-11). These two types of base pairs predominate in double-strandedDNA and RNA, and the tautomers shown in Figure 12-2 are responsible for these patterns.
This specific pairing of bases permits the duplication of genetic information by the synthesis of nucleic acid strandsthat are complementary to existing strands, as we shall discuss later inthis chapter.3"Nucleic Acid StructureThe discovery of the structure of DNA by Watson and Crick in 1953 wasa momentous event in science, an event that gave rise to entirely newdisciplines and influenced the course of many others.
Our present understanding of the storage and utilization of a cell's genetic information is based on work made possible by this discovery. Although information pathways are not treated in detail until Part IV of this book,the outline of these pathways presented in Chapters 1 and 3 is now aprerequisite for discussion of any area of biochemistry. Here, we concern ourselves with DNA structure itself, events that led to its discovery, and more recent refinements in our understanding.
RNA structurewill also be introduced.As in the case of protein structure (Chapters 6 and 7), it is sometimes useful to describe nucleic acid structure in terms of hierarchicallevels of complexity (primary, secondary, tertiary). The primary structure of a nucleic acid is its covalent structure and nucleotide sequence.Any regular, stable structure taken up by some or all of the nucleotidesin a nucleic acid can be referred to as secondary structure. All of thestructures considered in the following pages of this chapter fall underthe heading of secondary structure.
The complex folding of large chromosomes within the bacterial nucleoid and eukaryotic chromatin isgenerally considered tertiary structure; this is considered inChapter 23.James WatsonFrancis CrickRDNA Stores Genetic InformationLive encapsulatedvirulent bacteriaMouse diesLive nonencapsulatednonvirulent bacteriaV Mouse lives(b)LiveencapsulatedvirulentHeat-killedbacteria virulent bacteriaMouse lives(c)Heat-killed virulent bacteriaLive nonencapsulatednonvirulentMixturebacteriaof heat-killedvirulent and livenonvirulent bacteriaMouse dies(d), Live nonvirulent bacteriaDNA isolated from heat-killedvirulent bacteriaLive nonencapsulatednonvirulentbacteriaEncapsulatedvirulent bacteria(e)Mouse diesThe biochemical investigation of DNA began with Friedrich Miescher,who carried out the first systematic chemical studies of cell nuclei.
In1868 Miescher isolated a phosphorus-containing substance, which hecalled "nuclein," from the nuclei of pus cells (leukocytes) obtained fromdiscarded surgical bandages. He found nuclein to consist of an acidicportion, which we know today as DNA, and a basic portion, protein.Miescher later found a similar acidic substance in the heads of salmonsperm cells. Although he partially purified the nucleic acid and studiedits properties, the covalent (primary) structure of DNA (as shown inFig.
12-7) did not become known with certainty until the late 1940s.Miescher and many others suspected that nuclein or nucleic acidwas associated in some way with cell inheritance, but the first directevidence that DNA is the bearer of genetic information came in 1944through a discovery made by Oswald T. Avery, Colin MacLeod, andMaclyn McCarty. These investigators found that DNA extracted from avirulent (disease-causing) strain of the bacterium Streptococcus pneumoniae, also known as pneumococcus, genetically transformed a nonvirulent strain of this organism into a virulent form (Fig. 12-12).
Averyand his colleagues concluded that the DNA extracted from the virulentstrain carried the inheritable genetic message for virulence. Not everyone accepted these conclusions, because traces of protein impuritiespresent in the DNA could have been the actual carrier of the geneticinformation. This possibility was soon eliminated by the finding thattreatment of the DNA with proteolytic enzymes did not destroy thetransforming activity, but treatment with deoxyribonucleases (DNAhydrolyzing enzymes) did.A second important experiment provided independent evidencethat DNA carries genetic information.
In 1952 Alfred D. Hershey andMartha Chase used radioactive phosphorus (32P) and radioactive sulfur (35S) tracers to show that when the bacterial virus (bacteriophage)T2 infects its host cell, E. coli, it is the phosphorus-containing DNA ofFigure 12-12 The Avery-MacLeod-McCarty experiment. When injected into mice, the encapsulated strain of pneumococcus (a) is lethal, whereasthe nonencapsulated strain (b) is harmless, as isthe heat-killed encapsulated strain (c). Earlier research by the bacteriologist Frederick Griffith hadshown that adding heat-killed virulent bacteria(which alone are harmless to mice) to a live nonvirulent strain permanently transformed the latterinto lethal, virulent, encapsulated bacteria (d).
Heconcluded that a transforming factor in the heatkilled virulent bacteria had gained entrance intothe live nonvirulent bacteria and rendered themvirulent and encapsulated.Avery and his colleagues identified the Griffithtransforming factor as DNA. (e) They extracted theDNA from heat-killed virulent pneumococci, removing the protein as completely as possible, and addedthis DNA to nonvirulent bacteria. The nonvirulentpneumococci were permanently transformed into avirulent strain. The DNA evidently gained entranceinto the nonvirulent bacteria, and the genes forvirulence and capsule formation became incorporated into the chromosomes of the nonvirulentbacteria. All subsequent generations of thesebacteria were therefore virulent and encaDsulated.Chapter 12 Nucleotides and Nucleic AcidsFigure 12—13 Summary of the Hershey—Chaseexperiment.
Two batches of isotopically labeled bacteriophage particles were prepared. One was labeled with 32P in the phosphate groups of the DNAand the other with 35S in the sulfur-containingamino acids of the protein coats (capsids). (Notethat DNA contains no sulfur, and viral protein nophosphorus.) The two batches of labeled phage werethen added to separate suspensions of unlabeledbacteria.
Each suspension of phage-infected cellswas agitated in a blender to shear the viral capsidsfrom the bacteria. The bacteria and empty viralcoats (ghosts) were then separated by centrifugation. The cells infected with the 32P-labeled phagewere found to contain 32P, indicating that the labeled viral DNA had entered the cells, and theviral ghosts contained no radioactivity. The cellsinfected with 35S-labeled phage were found to haveno radioactivity after blender treatment, but theviral ghosts contained 35S.
Progeny virus particleswere produced in both batches of bacteria sometime after the viral coats were removed, thus thegenetic message for their replication had been introduced by viral DNA, not by viral protein.32P experiment333S experimentNonradioactivecoatRadioactivecoatRadioactiveDNABlendertreatmentshears offviral headsthe viral particle, not the sulfur-containing protein of the viral coat,that actually enters the host cell and furnishes the genetic informationfor viral replication (Fig.
12-13).These important early experiments and many other lines of evidence have shown that DNA is definitely the exclusive chromosomalcomponent bearing the genetic information of living cells.DNAs Have Distinctive Base CompositionsA most important clue to the structure of DNA came from the work ofErwin Chargaff and his colleagues in the late 1940s. They found thatthe four nucleotide bases in DNA occur in different ratios in the DNAsof different organisms and that the amounts of certain bases areclosely related. These data, collected from DNAs of a great many different species, led Chargaff to the following conclusions:1. The base composition of DNA generally varies from one species toanother.2. DNA specimens isolated from different tissues of the same specieshave the same base composition.3.
The base composition of DNA in a given species does not changewith the organism's age, nutritional state, or changing environment.4. In all DNAs, regardless of the species, the number of adenine residues is equal to the number of thymine residues (that is, A = T),and the number of guanine residues is equal to the number of cytosine residues (G = C). From these relationships it follows that thesum of the purine residues equals the sum of the pyrimidine residues; that is, A + G = T + C.These quantitative relationships, sometimes called "ChargafFsrules," were confirmed by many subsequent researchers.
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