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`LCY Biotechnology Holding, Inc.
`Ex. 1007
`Page 2 of 22
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`Molecular Biology
`
`Fifth Edition
`
`R o b e r t F . W e a v e r
`University of Kansas
`
`TM
`
`LCY Biotechnology Holding, Inc.
`Ex. 1007
`Page 3 of 22
`
`

`

`TM
`
`MOLECULAR BIOLOGY, FIFTH EDITION
`
`Published by McGraw-Hill, a business unit of The McGraw-Hill Companies, Inc., 1221 Avenue of the
`Americas, New York, NY 10020. Copyright © 2012 by The McGraw-Hill Companies, Inc. All rights reserved.
`Previous editions © 2008, 2005, and 2002. No part of this publication may be reproduced or distributed in
`any form or by any means, or stored in a database or retrieval system, without the prior written consent of
`The McGraw-Hill Companies, Inc., including, but not limited to, in any network or other electronic storage or
`transmission, or broadcast for distance learning.
`
`Some ancillaries, including electronic and print components, may not be available to customers outside the
`United States.
`
`This book is printed on acid-free paper.
`
`1 2 3 4 5 6 7 8 9 0 QDB /QDB 1 0 9 8 7 6 5 4 3 2 1
`
`ISBN 978-0-07-352532-7
`MHID 0-07-352532-4
`
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`copyright page.
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`Library of Congress Cataloging-in-Publication Data
`
`Weaver, Robert Franklin, 1942-
`Molecular biology / Robert F. Weaver.—5th ed.
`p. cm.
`ISBN 978–0–07–352532–7 (hardcover : alk. paper)
`1. Molecular biology. I. Title.
`QH506.W43 2011
`572.8—dc22
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`LCY Biotechnology Holding, Inc.
`Ex. 1007
`Page 4 of 22
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`wea25324_ch02_012-029.indd Page 12 11/9/10 3:00 PM user-f468
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`/Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefiles
`
`C H A P T E R
`
`2
`
`The Molecular Nature of Genes
`
` Before we begin to study in detail
`
`the structure and activities of genes, and the
`experimental evidence underlying those
`concepts, we need a fuller outline of the
`adventure that lies before us. Thus, in this
`chapter and in Chapter 3, we will fl esh out
`the brief history of molecular biology pre-
`sented in Chapter 1. In this chapter we will
`begin this task by considering the behavior
`of genes as molecules.
`
`Computer model of the DNA double helix.
`© Comstock Images/Jupiter RF.
`
`LCY Biotechnology Holding, Inc.
`Ex. 1007
`Page 5 of 22
`
`

`

`2.1 The Nature of Genetic
`Material
`
`The studies that eventually revealed the chemistry of
`genes began in T übingen, Germany, in 1869. There, Friedrich
`Miescher isolated nuclei from pus cells (white blood cells)
`in waste surgical bandages. He found that these nuclei
`contained a novel phosphorus-bearing substance that he
`named nuclein. Nuclein is mostly chromatin, which is a
`complex of deoxyribonucleic acid (DNA) and chromoso-
`mal proteins.
` By the end of the nineteenth century, both DNA and
` ribonucleic acid (RNA) had been separated from the pro-
`tein that clings to them in the cell. This allowed more de-
`tailed chemical analysis of these nucleic acids. (Notice that
`the term nucleic acid and its derivatives, DNA and RNA,
`come directly from Miescher ’s term nuclein. ) By the begin-
`ning of the 1930s, P. Levene, W. Jacobs, and others had
`demonstrated that RNA is composed of a sugar (ribose)
`plus four nitrogen-containing bases, and that DNA con-
`tains a different sugar (deoxyribose) plus four bases. They
`discovered that each base is coupled with a sugar –phosphate
`to form a nucleotide. We will return to the chemical struc-
`tures of DNA and RNA later in this chapter. First, let us
`examine the evidence that genes are made of DNA.
`
`Transformation in Bacteria
`Frederick Griffi th laid the foundation for the identifi cation
`of DNA as the genetic material in 1928 with his experi-
`ments on transformation in the bacterium pneumococcus,
`now known as Streptococcus pneumoniae. The wild-type
`organism is a spherical cell surrounded by a mucous coat
`called a capsule. The cells form large, glistening colonies,
`characterized as smooth (S) ( Figure 2.1a ). These cells are
` virulent, that is, capable of causing lethal infections upon
`injection into mice. A certain mutant strain of S. pneu-
`moniae has lost the ability to form a capsule. As a result, it
`grows as small, rough (R) colonies ( Figure 2.1b ). More im-
`portantly, it is avirulent; because it has no protective coat, it
`is engulfed by the host ’s white blood cells before it can pro-
`liferate enough to do any damage.
` The key fi nding of Griffi th ’s work was that heat-killed
`virulent colonies of S. pneumoniae could transform aviru-
`lent cells to virulent ones. Neither the heat-killed virulent
`bacteria nor the live avirulent ones by themselves could
`cause a lethal infection. Together, however, they were
`deadly. Somehow the virulent trait passed from the dead
`cells to the live, avirulent ones. This transformation phe-
`nomenon is illustrated in Figure 2.2 . Transformation was
`not transient; the ability to make a capsule and therefore to
`kill host animals, once conferred on the avirulent bacteria,
`was passed to their descendants as a heritable trait. In other
`words, the avirulent cells somehow gained the gene for
`
`2.1 The Nature of Genetic Material
`
` 13
`
`(a)
`
`(b)
`
`Figure 2.1 Variants of Streptococcus pneumoniae: (a) The large,
`glossy colonies contain smooth (S) virulent bacteria; (b) the small,
`mottled colonies are composed of rough (R) avirulent bacteria.
` (Source: (a, b) Harriet Ephrussi-Taylor.)
`
` virulence during transformation. This meant that the trans-
`forming substance in the heat-killed bacteria was probably
`the gene for virulence itself. The missing piece of the puzzle
`was the chemical nature of the transforming substance.
`
`DNA: The Transforming Material Oswald Avery, Colin
`MacLeod, and Maclyn McCarty supplied the missing
`piece in 1944. They used a transformation test similar to
`the one that Griffi th had introduced, and they took pains
`to defi ne the chemical nature of the transforming sub-
`stance from virulent cells. First, they removed the protein
`from the extract with organic solvents and found that the
`extract still transformed. Next, they subjected it to diges-
`tion with various enzymes. Trypsin and chymotrypsin,
`which destroy protein, had no effect on transformation.
`Neither did ribonuclease, which degrades RNA. These
` experiments ruled out protein or RNA as the transforming
`material. On the other hand, Avery and his coworkers
`found that the enzyme deoxyribonuclease (DNase), which
`breaks down DNA, destroyed the transforming ability of
`the virulent cell extract. These results suggested that the
`transforming substance was DNA.
` Direct physical-chemical analysis supported the hypo-
`thesis that the purifi ed transforming substance was DNA.
`The analytical tools Avery and his colleagues used were
`the following:
`
`1.
`
` Ultracentrifugation They spun the transforming
` substance in an ultracentrifuge (a very high-speed
`
`LCY Biotechnology Holding, Inc.
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`14
`
` Chapter 2 / The Molecular Nature of Genes
`
`Strain of
`Colony
`
`Strain of
`Colony
`
`Cell type
`
`Effect
`
`– Capsule
`
`Cell type
`
`No capsule
`
`Effect
`
`Smooth (S)
`
`Live S
`strain
`
`(a)
`
`Rough (R)
`
`Live R
`strain
`
`(b)
`
`Effect
`
`Live R strain
`
`Effect
`
`Heat-killed
`S strain
`
`(c)
`
`Heat-killed
`S strain
`
`(d)
`
`Live S and R strains
`isolated from dead
`mouse
`
`Figure 2.2 Griffi th ’s transformation experiments. (a) Virulent strain S S. pneumoniae bacteria kill their host;
` (b) avirulent strain R bacteria cannot infect successfully, so the mouse survives; (c) strain S bacteria that are
`heat-killed can no longer infect; (d) a mixture of strain R and heat-killed strain S bacteria kills the mouse. The
`killed virulent (S) bacteria have transformed the avirulent (R) bacteria to virulent (S).
`
`centrifuge) to estimate its size. The material with
`transforming activity sedimented rapidly (moved
` rapidly toward the bottom of the centrifuge tube),
`suggesting a very high molecular weight, characteris-
`tic of DNA.
` Electrophoresis They placed the transforming
` substance in an electric fi eld to see how rapidly it
`moved. The transforming activity had a relatively high
`mobility, also characteristic of DNA because of its
`high charge-to-mass ratio.
` Ultraviolet Absorption Spectrophotometry They
`placed a solution of the transforming substance in a
`spectrophotometer to see what kind of ultraviolet
`(UV) light it absorbed most strongly. Its absorption
`spectrum matched that of DNA. That is, the light it
`
`2.
`
`3.
`
`4.
`
`absorbed most strongly had a wavelength of about
`260 nanometers (nm), in contrast to protein, which
`absorbs maximally at 280 nm.
` Elementary Chemical Analysis This yielded an
` average nitrogen-to-phosphorus ratio of 1.67,
`about what one would expect for DNA, which is
`rich in both elements, but vastly lower than the
`value expected for protein, which is rich in nitrogen
`but poor in phosphorus. Even a slight protein
` contamination would have raised the nitrogen-to-
`phosphorus ratio.
`
`Further Confi rmation These fi ndings should have settled
`the issue of the nature of the gene, but they had little imme-
`diate effect. The mistaken notion, from early chemical
`
`LCY Biotechnology Holding, Inc.
`Ex. 1007
`Page 7 of 22
`
`

`

`analyses, that DNA was a monotonous repeat of a four-
`nucleotide sequence, such as ACTG-ACTG-ACTG, and so
`on, persuaded many geneticists that it could not be the
`genetic material. Furthermore, controversy persisted about
`possible protein contamination in the transforming mate-
`rial, whether transformation could be accomplished with
`other genes besides those governing R and S, and even
`whether bacterial genes were like the genes of higher
` organisms.
` Yet, by 1953, when James Watson and Francis Crick
`published the double-helical model of DNA structure,
`most geneticists agreed that genes were made of DNA.
`What had changed? For one thing, Erwin Chargaff had
`shown in 1950 that the bases were not really found in
`equal proportions in DNA, as previous evidence had sug-
`gested, and that the base composition of DNA varied
`from one species to another. In fact, this is exactly what
`one would expect for genes, which also vary from one
`species to another. Furthermore, Rollin Hotchkiss had
`refi ned and extended Avery ’s fi ndings. He purifi ed the
`transforming substance to the point where it contained
`only 0.02% protein and showed that it could still change
`the genetic characteristics of bacterial cells. He went on
`to show that such highly purifi ed DNA could transfer
`genetic traits other than R and S.
`
`Finally, in 1952, A. D. Hershey and Martha Chase per-
`formed another experiment that added to the weight of
`evidence that genes were made of DNA. This experiment
`involved a bacteriophage (bacterial virus) called T2
`that infects the bacterium Escherichia coli ( Figure 2.3 ).
`
`Figure 2.3 A false color transmission electron micrograph of T2
`phages infecting an E. coli cell. Phage particles at left and top
`appear ready to inject their DNA into the host cell. Another T2 phage
`has already infected the cell, however, and progeny phage particles
`are being assembled. The progeny phage heads are readily discernible
`as dark polygons inside the host cell. (Source: © Lee Simon/Photo
`Researchers, Inc.)
`
`2.1 The Nature of Genetic Material
`
` 15
`
`(The term bacteriophage is usually shortened to phage. )
` During infection, the phage genes enter the host cell and
`direct the synthesis of new phage particles. The phage is
`composed of protein and DNA only. The question is
`this: Do the genes reside in the protein or in the DNA?
`The Hershey –Chase experiment answered this question
`by showing that, on infection, most of the DNA entered
`the bacterium, along with only a little protein. The bulk
`of the protein stayed on the outside ( Figure 2.4 ). Be-
`cause DNA was the major component that got into the
`host cells, it likely contained the genes. Of course, this
`conclusion was not unequivocal; the small amount of
`protein that entered along with the DNA could conceiv-
`ably have carried the genes. But taken together with the
`work that had gone before, this study helped convince
`geneticists that DNA, and not protein, is the genetic
`material.
` The Hershey –Chase experiment depended on radioac-
`tive labels on the DNA and protein —a different label for
`each. The labels used were phosphorus-32 ( 32 P) for DNA
`and sulfur-35 ( 35 S) for protein. These choices make sense,
`considering that DNA is rich in phosphorus but phage
`protein has none, and that protein contains sulfur but
`DNA does not.
` Hershey and Chase allowed the labeled phages to
`attach by their tails to bacteria and inject their genes
`into their hosts. Then they removed the empty phage
`coats by mixing vigorously in a blender. Because they
`knew that the genes must go into the cell, their ques-
`tion was: What went in, the 32 P-labeled DNA or the
` 35 S-labeled protein? As we have seen, it was the
`DNA.  In general, then, genes are made of DNA. On
`the  other hand, as we will see later in this chapter,
`other experiments showed that some viral genes consist
`of RNA.
`
`SUMMARY Physical-chemical experiments involv-
`ing bacteria and a bacteriophage showed that their
`genes are made of DNA.
`
`The Chemical Nature of Polynucleotides
`By the mid-1940s, biochemists knew the fundamental
`chemical structures of DNA and RNA. When they broke
`DNA into its component parts, they found these con-
`stituents to be nitrogenous bases, phosphoric acid, and
`the sugar deoxyribose (hence the name deoxyribonucleic
`acid ). Similarly, RNA yielded bases and phosphoric acid,
`plus a different sugar, ribose. The four bases found in
`DNA are adenine (A), cytosine (C), guanine (G), and
` thymine (T). RNA contains the same bases, except that
` uracil (U) replaces thymine. The structures of these bases,
`
`LCY Biotechnology Holding, Inc.
`Ex. 1007
`Page 8 of 22
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`

`

`16
`
` Chapter 2 / The Molecular Nature of Genes
`
`Protein coat is
`labeled specifically
`with 35S
`
`DNA is labeled
`specifically with 32P
`
`Attachment of phage
`to host cells
`
`Attachment of phage
`to host cells
`
`Removal of phage
`coats by blending
`
`Removal of phage
`coats by blending
`
`Cell containing little
`35S-labeled protein,
`plus unlabeled DNA
`
`Cell containing
`32P-labeled DNA
`
`(a)
`
`(b)
`
`Figure 2.4 The Hershey —Chase experiment. Phage T2 contains
`genes that allow it to replicate in E. coli. Because the phage is
`composed of DNA and protein only, its genes must be made of
`one of these substances. To discover which, Hershey and Chase
`performed a two-part experiment. In the fi rst part (a), they labeled
`the phage protein with 35 S (red), leaving the DNA unlabeled (black).
`In the second part (b), they labeled the phage DNA with 32 P (red),
`
`leaving the protein unlabeled (black). Since the phage genes must
`enter the cell, the experimenters reasoned that the type of label
`found in the infected cells would indicate the nature of the genes.
`Most of the labeled protein remained on the outside and was
`stripped off the cells by use of a blender (a), whereas most of the
`labeled DNA entered the infected cells (b). The conclusion was that
`the genes of this phage are made of DNA.
`
`shown in Figure 2.5 , reveal that adenine and guanine are
`related to the parent molecule, purine. Therefore, we
`refer to these compounds as purines. The other bases
`resemble pyrimidine, so they are called pyrimidines.
`These structures constitute the alphabet of genetics.
`
` Figure 2.6 depicts the structures of the sugars found in
`nucleic acids. Notice that they differ in only one place.
`Where ribose contains a hydroxyl (OH) group in the
`2-position, deoxyribose lacks the oxygen and simply has
`a hydrogen (H), represented by the vertical line. Hence
`the name deoxyribose. The bases and sugars in RNA and
`DNA are joined together into units called nucleosides
`( Figure 2.7 ). The names of the nucleosides derive from the
`corresponding bases:
`
` Base
`Adenine
`Guanine
`Cytosine
`Uracil
`Thymine
`
` Nucleoside (RNA)
` Adenosine
` Guanosine
` Cytidine
` Uridine
` Not usually found
`
` Deoxynucleoside (DNA)
` Deoxyadenosine
` Deoxyguanosine
` Deoxycytidine
` Not usually found
` (Deoxy)thymidine
`
` Because thymine is not usually found in RNA, the
` “deoxy ” designation for its nucleoside is frequently
` assumed, and the deoxynucleoside is simply called
` thymidine. The numbering of the carbon atoms in the
`sugars of the nucleosides (see Figure 2.7 ) is important.
`Note that the ordinary numbers are used in the bases, so
`
`LCY Biotechnology Holding, Inc.
`Ex. 1007
`Page 9 of 22
`
`

`

`2.1 The Nature of Genetic Material
`
` 17
`
`C–H
`
`N
`
`N
`H
`
`NH2
`C
`
`N
`
`C C
`
`OH
`
`CH2OH
`O
`
`C
`
`C
`
`N1
`
`2
`
`6
`
`7
`N
`
`5
`
`N
`H
`
`9
`
`4
`
`N
`3
`Purine
`
`4
`
`N
`
`8
`
`NH2
`
`N
`
`N
`H
`
`N
`
`Adenine
`
`NH2
`
`O
`
`O
`
`HN
`
`H2N
`
`N
`
`Guanine
`
`O
`
`3N
`
`N
`
`HN
`
`HN
`
`N
`
`N
`H
`
`CH3
`
`N
`
`H–C
`
`NH2
`
`N
`
`(a)
`
`N
`
`N
`
`N
`H
`
`OH
`
`CH2OH
`O
`
`H
`
`H H
`
`C
`
`H
`C
`
`H
`
`OH
`
`O
`
`N
`H
`Cytosine
`
`O
`
`N
`H
`Uracil
`
`O
`
`N
`H
`Thymine
`
`OH
`
`(b)
`
`5 6
`
`N 1
`
`2
`
`Pyrimidine
`
`Figure 2.5 The bases of DNA and RNA. The parent bases, purine
`and pyrimidine, on the left, are not found in DNA and RNA. They are
`shown for comparison with the other fi ve bases.
`
`Figure 2.8 The structures of (a) adenine and (b) deoxyribose.
`Note that the structures on the left do not designate most or all of
`the carbons and some of the hydrogens. These designations are
`included in the structures on the right, in red and blue, respectively.
`
` adenine and deoxyribose, fi rst in shorthand, then with
`every atom included.
` The subunits of DNA and RNA are nucleotides, which
`are nucleosides with a phosphate group attached through
`a phosphoester bond ( Figure 2.9 ). An ester is an organic
`compound formed from an alcohol (bearing a hydroxyl
`group) and an acid. In the case of a nucleotide, the alcohol
`group is the 5 9-hydroxyl group of the sugar, and the acid
`is phosphoric acid, which is why we call the ester a phos-
`phoester. Figure 2.9 also shows the structure of one of the
`four DNA precursors, deoxyadenosine-5 9-triphosphate
`(dATP). When synthesis of DNA takes place, two phos-
`phate groups are removed from dATP, leaving deoxy-
`adenosine-5 9-monophosphate (dAMP). The other three
`nucleotides in DNA (dCMP, dGMP, and dTMP) have
`analogous structures and names.
` We will discuss the synthesis of DNA in detail in
`Chapters 20 and 21. For now, notice the structure of the
`bonds that join nucleotides together in DNA and RNA
`( Figure 2.10 ). These are called phosphodiester bonds
` because they involve phosphoric acid linked to two
` sugars: one through a sugar 5 9-group, the other through
`a sugar 3 9-group. You will notice that the bases have
`been rotated in this picture, relative to their positions in
`previous fi gures. This more closely resembles their geo-
`metry in DNA or RNA. Note also that this trinucleotide,
`or string of three nucleotides, has polarity: The top of
`the molecule bears a free 5 9-phosphate group, so it is
`called the 5 9-end. The bottom, with a free 3 9-hydroxyl
`group, is called the 3 9-end.
`
` Figure 2.11 introduces a shorthand way of represent-
`ing a nucleotide or a DNA chain. This notation presents
`the deoxyribose sugar as a vertical line, with the base
`joined to the 1 9-position at the top and the phospho-
`diester links to neighboring nucleotides through the
`3 9-(middle) and 5 9-(bottom) positions.
`
`CH2OH
`O
`
`OH
`
`OH
`2-deoxyribose
`
`OH
`
`1
`
`CH2OH
`O
`
`5 4
`
`3
`2
`OH
`OH
`Ribose
`
`Figure 2.6 The sugars of nucleic acids. Note the OH in the
`2-position of ribose and its absence in deoxyribose.
`
`NH2
`
`N
`
`N
`
`CH2OH
`5፱
`O
`
`4፱
`
`N
`
`N
`
`1፱
`
`3፱
`OH
`
`2፱
`OH
`
`Adenosine
`
`O
`
`HN
`
`CH3
`
`O
`
`N
`
`CH2OH
`5፱
`O
`
`4፱
`
`3፱
`OH
`
`1፱
`
`2፱
`
`2፱-deoxythymidine
`
`Figure 2.7 Two examples of nucleosides.
`
`the carbons in the sugars are called by primed numbers.
`Thus, for example, the base is linked to the 1 9-position
`of the sugar, the 2 9-position is deoxy in deoxynucleosides,
`and the sugars are linked together in DNA and RNA
`through their 3 9- and 5 9-positions.
` The structures in Figure 2.5 were drawn using an
` organic chemistry shorthand that leaves out certain
` atoms for simplicity ’s sake. Figures 2.6 and 2.7 use a
`slightly different convention, in which a straight line
`with a free end denotes a C–H bond with a hydrogen
`atom at the end. Figure 2.8 shows the structures of
`
`LCY Biotechnology Holding, Inc.
`Ex. 1007
`Page 10 of 22
`
`

`

`18
`
` Chapter 2 / The Molecular Nature of Genes
`
`N
`
`N
`
`NH2
`
`N
`
`N
`
`CH2
`
`O
`
`O
`
`OPO
`O–
`α
`
`PO
`
`O–
`β
`
`O
`
`OP
`O–
`γ
`
`–O
`
`N
`
`N
`
`NH2
`
`N
`
`N
`
`CH2
`
`O
`
`O
`
`OPO
`O–
`
`PO
`
`O–
`
`–O
`
`NH2
`
`N
`
`–O
`
`O
`
`OP
`O–
`
`N
`
`CH2
`
`O
`
`N
`
`N
`
`OH
`Deoxyadenosine-5፱-
`monophosphate (dAMP)
`
`OH
`Deoxyadenosine-5፱-
`diphosphate (dADP)
`
`OH
`Deoxyadenosine-5፱-
`triphosphate (dATP)
`
`Figure 2.9 Three nucleotides. The 5 9-nucleotides of deoxyadenosine are formed by phosphorylating the
`5 9-hydroxyl group. The addition of one phosphate results in deoxyadenosine-5 9-monophosphate (dAMP).
`One more phosphate yields deoxyadenosine-5 9-diphosphate (dADP). Three phosphates (designated
` a, b, g) give deoxyadenosine-5 9-triphosphate (dATP).
`
`O
`
`H3C
`
`A
`1′
`
`T
`1′
`
`C
`1′
`
`A
`1′
`
`3′
`
`3′
`
`3′
`
`3′
`
`P
`
`P
`
`P
`
`OH
`
`P
`
`P
`
`P
`
`OH
`
`(a) dATP
`
`5′
`
`5′
`5′
`(b) DNA strand
`
`5′
`
`Figure 2.11 Shorthand DNA notation. (a) The nucleotide dATP.
`This illustration highlights four features of this DNA building block:
`(1) The deoxyribose sugar is represented by the vertical black line.
`(2) At the top, attached to the 1 9-position of the sugar is the base,
`adenine (green). (3) In the middle, at the 3 9-position of the sugar is
`a hydroxyl group (OH, orange). (4) At the bottom, attached to the
`5 9-position of the sugar is a triphosphate group (purple). (b) A short DNA
`strand. The same trinucleotide (TCA) illustrated in Figure 2.10 is shown
`here in shorthand. Note the 5 9-phosphate and the phosphodiester bonds
`(purple), and the 3 9-hydroxyl group (orange). According to convention,
`this little piece of DNA is written 5 9 to 3 9 left to right.
`
`SUMMARY DNA and RNA are chain-like mole-
`cules composed of subunits called nucleotides.
`The nucleotides contain a base linked to the
` 1 9-position of a sugar (ribose in RNA or deoxyri-
`bose in DNA) and a phosphate group. The phos-
`phate joins the sugars in a DNA or RNA chain
`through their 5 9- and 3 9-hydroxyl groups by phos-
`phodiester bonds.
`
`NH
`
`(T)
`
`N
`
`O
`
`5፱-phosphate
`
`O
`
`CH2
`
`O
`
`–
`
`O P O
`
`–O
`
`NH2
`
`N
`
`(C)
`
`O
`
`N
`
`O
`
`OPO
`O–
`
`CH2
`
`O
`
`NH2
`
`N
`
`N
`
`N
`
`N
`
`(A)
`
`Phosphodiester
`bonds
`
`O
`
`OPO
`O–
`
`CH2
`
`O
`
`3፱-hydroxyl
`
`OH
`
`Figure 2.10 A trinucleotide. This little piece of DNA contains only
`three nucleotides linked together by phosphodiester bonds (red)
`between the 5 9- and 3 9-hydroxyl groups of the sugars. The 5 9-end
`of this DNA is at the top, where a free 5 9-phosphate group (blue) is
`located; the 3 9-end is at the bottom, where a free 3 9-hydroxyl group
`(also blue) appears. The sequence of this DNA could be read as
`5 9pdTpdCpdA3 9. This would usually be simplifi ed to TCA.
`
`2.2 DNA Structure
`
`All the facts about DNA and RNA just mentioned were
`known by the end of the 1940s. By that time it was also
`becoming clear that DNA was the genetic material and
`
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`

`

`that it therefore stood at the very center of the study of
`life. Yet the three-dimensional structure of DNA was un-
`known. For these reasons, several researchers dedicated
`themselves to fi nding this structure.
`
`Experimental Background
`One of the scientists interested in DNA structure was Linus
`Pauling, a theoretical chemist at the California Institute of
`Technology. He was already famous for his studies on
`chemical bonding and for his elucidation of the a-helix; an
`important feature of protein structure. Indeed, the a-helix,
`held together by hydrogen bonds, laid the intellectual
`groundwork for the double-helix model of DNA proposed
`by Watson and Crick. Another group trying to fi nd the
`structure of DNA included Maurice Wilkins, Rosalind
`Franklin, and their colleagues at King ’s College in London.
`They were using x-ray diffraction to analyze the three-
`dimensional structure of DNA. Finally, James Watson and
`Francis Crick entered the race. Watson, still in his early
`twenties, but already holding a Ph.D. degree from Indiana
`University, had come to the Cavendish Laboratories in
`Cambridge, England, to learn about DNA. There he met
`Crick, a physicist who at age 35 was retraining as a mo-
`lecular biologist. Watson and Crick performed no experi-
`ments themselves. Their tactic was to use other groups ’
`data to build a DNA model.
` Erwin Chargaff was another very important contribu-
`tor. We have already seen how his 1950 paper helped
`identify DNA as the genetic material, but the paper con-
`tained another piece of information that was even more
`signifi cant. Chargaff ’s studies of the base compositions of
`DNAs from various sources revealed that the content of
`purines was always roughly equal to the content of py-
`rimidines. Furthermore, the amounts of adenine and thy-
`mine were always roughly equal, as were the amounts of
`guanine and cytosine. These fi ndings, known as Char-
`gaff ’s rules, provided a valuable foundation for Watson
`and Crick ’s model. Table 2.1 presents Chargaff ’s data.
`
`2.2 DNA Structure
`
` 19
`
`You will notice some deviation from the rules due to in-
`complete recovery of some of the bases, but the overall
`pattern is clear.
`
`Perhaps the most crucial piece of the puzzle came
`from an x-ray diffraction picture of DNA taken by
`Franklin in 1952 —a picture that Wilkins shared with
`James Watson in London on January 30, 1953. The x-ray
`technique worked as follows: The experimenter made a
`very concentrated, viscous solution of DNA, then reached
`in with a needle and pulled out a fi ber. This was not a
`single molecule, but a whole batch of DNA molecules,
`forced into side-by-side alignment by the pulling action.
`Given the right relative humidity in the surrounding air,
`this fi ber was enough like a crystal that it diffracted
` x-rays in an interpretable way. In fact, the x-ray diffrac-
`tion pattern in Franklin ’s picture ( Figure 2.12 ) was so
`simple —a series of spots arranged in an X shape —that it
`indicated that the DNA structure itself must be very sim-
`ple. By contrast, a complex, irregular molecule like a pro-
`tein gives a complex x-ray diffraction pattern with many
`spots, rather like a surface peppered by a shotgun blast.
`Because DNA is very large, it can be simple only if it has
`a regular, repeating structure. And the simplest repeating
`shape that a long, thin molecule can assume is a
` corkscrew, or helix.
`
`The Double Helix
`Franklin ’s x-ray work strongly suggested that DNA was
`a helix. Not only that, it gave some important informa-
`tion about the size and shape of the helix. In particular,
`the spacing between adjacent bands in an arm of the X
`is inversely related to the overall repeat distance in the
`helix, 33.2 angstroms (33.2 Å), and the spacing from the
`top of the X to the bottom is inversely related to the spac-
`ing (3.32 Å) between the repeated elements ( base pairs )
`in the helix. (See Chapter 9 for information on how
`Bragg ’s law explains these inverse relationships.) How-
`ever, even though the Franklin picture told much about
`
`Table 2.1 Composition of DNA in Moles of Base per Mole of Phosphate
`
`Human
`
`Sperm
`
` Liver
` Thymus Carcinoma
`
`
`
` Yeast
`
` Avian
`Tubercle
`Bacilli
`
`A:
`T:
`G:
`C:
`Recovery:
`
`#1
`
` 0.29
` 0.31
` 0.18
` 0.18
` 0.96
`
` #2
`
` 0.27
` 0.30
` 0.17
` 0.18
` 0.92
`
` 0.28
` 0.28
` 0.19
` 0.16
` 0.91
`
` #1
`
` 0.24
` 0.25
` 0.14
` 0.13
` 0.76
`
` #2
`
` 0.30
` 0.29
` 0.18
` 0.15
` 0.92
`
` 0.12
` 0.11
` 0.28
` 0.26
` 0.77
`
` 0.27
` 0.27
` 0.18
` 0.15
` 0.87
`
` Bovine
`
` Thymus
`
` #1
`
` 0.26
` 0.25
` 0.21
` 0.16
` 0.88
`
` #2
`
` 0.28
` 0.24
` 0.24
` 0.18
` 0.94
`
` #3
`
` 0.30
` 0.25
` 0.22
` 0.17
` 0.94
`
` Spleen
`
` #1
`
` #2
`
` 0.25
` 0.24
` 0.20
` 0.15
` 0.84
`
` 0.26
` 0.24
` 0.21
` 0.17
` 0.88
`
` Source: E. Chargaff “Chemical Specifi city of Nucleic Acids and Mechanism of Their Enzymatic Degradation, ” Experientia 6:206, 1950.
`
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`

`20
`
` Chapter 2 / The Molecular Nature of Genes
`
`Figure 2.12 Franklin ’s x-ray picture of DNA. The regularity of this
`pattern indicated that DNA is a helix. The spacing between the
`bands at the top and bottom of the X gave the spacing between
`elements of the helix (base pairs) as 3.32 Å. The spacing between
`neighboring bands in the pattern gave the overall repeat of the helix
`(the length of one helical turn) as 33.2 Å. (Source: Courtesy Professor
`M.H.F. WIlkins, Biophysics Dept., King ’s College, London.)
`
`DNA, it presented a paradox: DNA was a helix with a
`regular, repeating structure, but for DNA to serve its
` genetic function, it must have an irregular sequence of
`bases.
` Watson and Crick saw a way to resolve this contra-
`diction and satisfy Chargaff ’s rules at the same time:
`DNA must be a double helix with its sugar –phosphate
`backbones on the outside and its bases on the inside.
`Moreover, the bases must be paired, with a purine in
`one strand always across from a pyrimidine in the
`other. This way the helix would be uniform; it would
`not have bulges where two large purines were paired or
`constrictions where two small pyrimidines were paired.
`Watson has joked about the reason he seized on a dou-
`ble helix: “I had decided to build two-chain models.
`Francis would have to agree. Even though he was a
`physicist, he knew that important biological objects
`come in pairs. ”
` But Chargaff ’s rules went further than this. They
`decreed that the amounts of adenine and thymine were
`equal and so were the amounts of guanine and cytosine.
`This fit very neatly with Watson and Crick ’s observa-
`tion that an adenine –thymine base pair held together by
`hydrogen bonds has almost exactly the same shape as a
`guanine –cytosine base pair ( Figure 2.13 ). So Watson
`and Crick postulated that adenine must always pair
`with thymine, and guanine with cytosine. This way, the
`double-stranded DNA will be uniform, composed of
`very similarly shaped base pairs, regardless of the
` unpredictable sequence of either DNA strand by itself.
`
`G
`
`N
`
`Sugar
`
`N
`
`O
`
`H
`
`N
`
`H
`
`HN
`
`HN
`
`N
`
`H
`
`H
`
`N
`
`N
`
`H
`
`N
`
`O
`
`O
`
`N
`
`Sugar
`
`CH3
`
`A
`
`N
`
`Sugar
`
`N
`
`N
`
`H
`
`N
`
`N
`
`O
`
`Sugar
`
`C
`
`T
`
`Figure 2.13 The base pairs of DNA. A guanine –cytosine pair (G –C),
`held together by three hydrogen bonds (dashed lines), has almost
`exactly the same shape as an adenine–thymine pair (A –T), held
`together by two hydrogen bonds.
`
`This was their crucial insight, and the key to the struc-
`ture of DNA.
` The double helix, often likened to a twisted ladder, is
`presented in three ways in Figure 2.14 . The curving sides
`of the ladder represent the sugar –phosphate backbones of
`the two DNA strands; the rungs are the base pairs. The
`spacing between base pairs is 3.32 Å, and the overall helix
`repeat distance is about 33.2 Å, meaning that there are
`about 10 base pairs ( bp ) per turn of the helix. (One
` angstrom [ Å] is one ten-billionth of a meter or one-tenth
`of a nanometer [nm].) The arrows indicate that the two
`strands are antiparallel. If one has 5 9 →3 9 polarity from
`top to bottom, the other must have 3 9 →5 9 polarity from
`top to bottom. In solution, DNA has a structure very sim-
`ilar to the one just described, but the helix contains about
`10.4 bp per turn.
` Watson and Crick published the outline of their
`model in the journal Nature, back-to-back with papers
`by Wilkins and Franklin and their coworkers showing
`the x-ray data. The Watson –Crick paper is a classic of
`simplicity —only 900 words, barely over a page long. It
`was published very rapidly, less than a month after it
`was submitted. Actually, Crick wanted to spell out the
`biological implications of the model, but Watson was
`uncomfortable doing that. They compromised on a sen-
`tence t