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The Source of Genetic

Information
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The Source of Genetic
Information
A. Malcolm Campbell, PhD

Christopher J. Paradise, PhD
The Source of Genetic Information

Copyright © A. Malcolm Campbell and Christopher J. Paradise. 2016.
All rights reserved. No part of this publication may be reproduced, stored

in a retrieval system, or transmitted in any form or by any means—
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brief quotations, not to exceed 250 words, without the prior permission
of the publisher.
First published in 2016 by

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Abstract
Everyone who has taken any biology class knows that DNA is the herita-
ble material. However, very few people know the evidence that led to this
conclusion. Science is a discipline based on evidence not acceptance based
on faith in a teacher or other authority. This book presents the historical
and scientific context to understand how we know DNA is the heritable
material. Furthermore, how the structure of DNA reveals its function
will be discussed. The famous double helix shape foretold how it would
be replicated. Two biochemists conducted the research to confirm that
each of the two strands serve as template for new DNA synthesis. Despite
its central role in cell function, the order of bases in DNA is not the full
story. This book also introduces the topic of epigenetics by presenting
the first animal experiments that showed epigenetic changes can lead to a
change in phenotype even though the DNA is not mutated.
Keywords
heritable material, emergent property, R colonies, S colonies, S factor,
DNA structure, nucleotides, deoxyribose, dNTP, purines, pyrimidines,
hydrogen bonds, covalent bonds, non-radioactive isotopes, semiconser
vative replication, methylated DNA, thin layer chromatography,
hypomethylated, hypermethylated, epigenetic, methyltransferase
Contents
Preface...................................................................................................ix
Acknowledgments....................................................................................xi
Introduction.........................................................................................xiii
Chapter 1 Defining Biological Information........................................1
Chapter 2 Search for the Heritable Information..................................3
Chapter 3 Disproving Proteins are the Heritable Information...........13
Ethical, Legal, Social Implications: DNA Ownership.......17
Chapter 4 DNA Structure Determines Its Function.........................21
The Structure of DNA.....................................................21
DNA Replication.............................................................27
Chapter 5 Not all DNA Information is Linear in Nature..................33
Conclusion............................................................................................39
Glossary................................................................................................41
Index....................................................................................................43
Preface
This book on the source of genetic material is part of a thirty book
series that collectively surveys all of the major themes in biology. Rather
than just present information as a collection of facts, the reader is treated
more like a scientist, which means the data behind the major themes are
presented. Reading any of the thirty books by Campbell and Paradise
provides readers with biological context and comprehensive perspective
so that readers can learn important information from a single book with
the potential to see how the major themes span all size scales: molecular,
cellular, organismal, population and ecologic systems. The major themes
of biology encapsulate the entire discipline: information, evolution, cells,
homeostasis and emergent properties.
In the twentieth century, biology was taught with a heavy emphasis
on long lists of terms and many specific details. All of these details were
presented in a way that obscured a more comprehensive understanding.
In this book, readers will learn how DNA was found to be the heritable
material and how its structure contributes to its function and some of the
supporting evidence behind our understanding. Instead of believing or
simply accepting information, readers of this book will learn about the
science behind the production of proteins the way professional scientists
do—with experimentation and data analysis. In short, data are put back
into the teaching of biological sciences.
Readers of this book who wish to see the textbook version of this
content can go to www.bio.davidson.edu/icb where they will find
pedagogically-designed and interactive Integrating Concepts in Biology for
introductory biology college courses or a high school AP Biology course.
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Acknowledgments
Publishing this book would not have been possible without the generous
gift of Dr. David Botstein who shared some of his Breakthrough Prize
with AMC. David’s gift allowed us to hire talented artists (Tom Webster
and his staff at Lineworks, Inc.) and copyeditor Laura Loveall. Thanks go
to Kristen Mandava for project management and guidance on the pub-
lishing process. In particular, we are indebted to Katie Noble and Melissa
Hayban for their many hours of help and attention to detail.
Kristen Eshleman, Paul Brantley, Bill Hatfield and Olivia Booker
helped us with technology at Davidson College. We are grateful to ad-
ministrators Tom Ross, Clark Ross, Carol Quillen, Wendy Raymond,
Verna Case, and Barbara Lom who had confidence in us and encouraged
us to persist despite setbacks along the way.
These books were the product of the shared labor of my two vision-
ary coauthors Laurie Heyer and Chris Paradise. We shared the dream
and the hardships and developed this book from scratch. My family has
been very supportive and I thank Susan, Celeste and Paulina for their
support and patience. I also want to thank Jan Serie, my pedagogical
mentor, who taught me so much about the art and science of helping stu-
dents learn. I benefited from the support of the Howard Hughes Medical
Institute grant 52006292, the James G. Martin Genomics Program, and
Davidson College. This book would not have survived its first draft with-
out my students who endured the typos and the early versions of this
book. These undergraduates participated in a bold experiment to see if
beginners could construct their own knowledge, retain what they learned,
and transform the way they see themselves and the discipline of biology.
While many people said that beginning students were not up to the task,
my students proved them wrong.
Introduction
What defines a human being? Why is every human different, even “iden-
tical” twins? A human body contains 10 to 50 trillion cells. Each cell
contains instructions for the processes and functions of a human body.
The information to carry out these functions is encoded in deoxyribo-
nucleic acid (DNA), half of which each person inherited from a mother
and a father. What evidence supports the claim that DNA is the heri-
table material? How does DNA relay its information to the next genera-
tion? For many years, scientists mistakenly thought that protein was the
heritable material. However, after many years of clever and now famous
experiments, the evidence mounted in favor of DNA and against protein
as the carrier of genetic information. One of the most famous discover-
ies in science was the double helix structure of DNA. DNA’s structure
helps explain how DNA replicates itself to produce the next generation.
Non-mutational chemical modifications to DNA can lead to differences
among individuals, including some we can observe and some that make
us sick. In this book, the reader will survey data from experiments that led
to our current understanding of DNA as heritable information. Genetic
information defines each person as a human being and differentiates each
person as an individual.
CHAPTER 1
Defining Biological
Information
Biology is the study of life. Students have learned this definition, but what
does life really mean? If anyone looks up the word life, Webster’s diction-
ary tells us life is the quality that distinguishes a vital functional being
from a dead body. In other words, life is not death. Frankly, this definition
of life is not very informative; biology is the study of a quality that is not
death. Most people mistakenly think of biology (and science in general)
as a compilation of facts or a vocabulary list. In reality, science is a pro-
cess of discovery based on observation and experimentation. Through the
science of biology in this book, the reader will discover information that
exists in nature and decipher information that characterizes the quality of
life. Professional biologists are not paid to memorize facts already discov-
ered. As a biologist, a student of life, each person’s charge is to discover
different layers of information represented in living organisms, regardless
of their size or complexity.
Looking up the word information in a dictionary will generate many
definitions, but two are particularly relevant: 1) communication or recep-
tion of knowledge; 2) signal representing data that justifies a change. One
aspect of biological information is the transmission of knowledge to other
living beings. Sharing ideas is commonplace for humans, and most people
consider the communication of knowledge to be a uniquely human trait.
But restricting information to human communication is a misconception.
Information can be conveyed between monkeys, dogs, bees, plants, preda-
tors and their prey, and even bacteria. A very famous example of infor-
mation as communicated knowledge is called “The Hundredth Monkey”
that became popularized in a book by the same name. In this 1950s study
of monkeys on a Japanese island, the investigators introduced a new food
2 THE SOURCE OF GENETIC INFORMATION
item, sweet potatoes, to see what the troop of monkeys would do. One
monkey used the ocean water to wash sand off of the potato, and the new
behavior spread to most of the young monkeys and a few of the older
adults. As the young monkeys began to reproduce, they taught their off-
spring, and the food washing behavior spread throughout the troop as
the older monkeys died out. The young monkeys began to use the ocean
for bathing and hunting seafood as well, which was an unexpected conse-
quence (i.e., an emergent property) of the learned behavior.
Information also means “signal representing data that justifies a
change.” Signals come in many forms and scales; short days means winter
season, a white stripe on a black fur coat warns other animals about a
skunk, bitter taste indicates poisonous food, chemical odors signal the
presence of a potential mate or predator, and loud sounds attract a mate
or deter an enemy. Much of life concerns detecting and interpreting sig-
nals produced by organisms, which is why information is so important in
biology. Biological information is transmitted throughout all life forms.
Signals stimulate a change in molecules, cells, organisms, populations, or
ecological systems that can receive and interpret the information. Perhaps
the most fundamental example of information transfer is reproduction
that is regulated at the molecular level.
Bibliography
Myers E: The hundredth monkey revisited: going back to the original
sources puts a new light on this popular story, Context Institute
(website): http://www.context.org/ICLIB/IC09/Myers.htm. Accessed
March 26, 2014.
CHAPTER 2
Search for the Heritable

Information
Science attempts to understand the physical world by answering ques-

tions through experimentation. For example, why do children often look
like their parents? Why do adults give rise to the same species? A critical
question in early biology research was, “What is the heritable material
that is passed from one generation to the next?”
Sometimes, great science arises from an astute observation and a simple
question. Dr. Fred Griffith was a medical officer in the British M
inistry of
Health during the 1920s. One of the major health threats at that time was
pneumonia caused by the bacterium Streptococcus pneumoniae. Between
1920 and 1927, Griffith collected samples from patients and performed
experiments on 278 isolates of pneumonia bacteria. At that time, the
way to diagnose pneumonia consisted of two procedures: 1) inject
a mouse with a patient’s saliva or mucus to see if the mouse died of pneu-
monia, and 2) spread the saliva or mucus on a Petri dish containing red
growth media and examine any colonies that grew.
While conducting these tests, Griffith noticed S. pneumoniae could
be classified into two strains, which he called rough (R) and smooth (S).
These two strains of bacteria could be distinguished visually by the shapes
of their colonies (rough or smooth). He first performed many experi-
ments to make sure he had not contaminated his samples. He also dis-
covered an interesting correlation: injecting bacteria from R colonies into
mice did not kill them, but injecting bacterial S colonies did. From all
his experimentation, he was convinced that S and R colonies were two
variants of the same species.
Based on his experiments and observations, Griffith hypothesized that
the harmless R cells could be converted to lethal S cells by an “S factor”
heat treatment heat treatment
S cells S cells R cells S cells

R cells
S cells No cells R cells S cells
Figure 1 Griffith’s experimental design and results. S cells and R cells

are labeled. Dead cells from heat treatment are indicated with “No”
symbol. Mice on their sides indicate those that died from pneumonia.
Arrows indicate progression through each part of the four experiments
shown in four columns.
Source: Original art based on data in Griffith, Fred. 1928. Griffith, Fred. 1928. The significance
of pneumococcal types. Journal of Hygene. 27: 113–159.
protein. To test his hypothesis, Griffith tried a series of experiments that

produced clear results (Figure 1). For his positive control, Griffith in-
jected S cells, which killed the mice as expected. Injecting R cells, which
did not kill the mice, served as his negative control. When the bacteria
were heated for an extended period of time, all of the cells were killed.
The most interesting experiment was when he co-injected heat-treated S
cells with live R cells.
Positive and negative controls are essential components of any good
experimental design. The purpose of controls is to verify that the experi-
ment has been performed properly. For example, R cells did not kill the
mice, but they did produce more R cells. This could be called a negative
control because the mice were not killed, or it could be called a positive
control because more R cells were produced. The name of the control
(positive or negative) is not the important aspect of experimental design.
The more important feature is that the investigator demonstrated the R
and S cells functioned as expected. Likewise, he demonstrated that heat is
sufficient to kill all S cells.
Search for the Heritable Information 5
Based on the results from the fourth experiment in Figure 1, Griffith

detected the existence of an “S factor” that was capable of transforming
R cells into S cells. Griffith later tested the new S cells in the last column
of Figure 1. The new S strain reproduced more S cells that had the same
morphology and virulence as the original S strain. Transformation from
R to S required an “S factor” to be released by the S cells and commu-
nicated to the R cells. At this point, Griffith wondered, “What is the
S factor?” Griffith hypothesized that the S factor was the heritable mate-
rial for all of life. This was bold speculation for a rather simple experiment.
All bacteria are composed of proteins, lipids, sugars, and nucleic acids,
and the S factor had to be one of these four categories of molecules. Pre-
vious research had ruled out lipids and carbohydrates as the heritable
material, so Griffith and many of the biologists of his generation were
convinced that protein, not DNA, was the heritable material. The reader
might remember that proteins are composed of twenty different amino
acids, whereas DNA and RNA are composed of only four different nucle-
otides. Therefore, proteins composed of twenty amino acids could encode
more information than a DNA sequence of the same length that is com-
posed of only four bases. Biologist in the 1920s used this mathematical
logic to mistakenly conclude protein was the heritable material instead
of DNA.
In the 1920s, it was common to separate bacterial cells into two
parts: cytoplasm and cell wall/membrane. The nucleic acids DNA and
ribonucleic acid (RNA) are located in the cytoplasm, whereas the cell
wall and membrane contain proteins and not nucleic acids. Because the
heritable material had been narrowed down to either nucleic acids or
protein, Griffith could design an experiment to characterize the S fac-
tor as protein (cell wall/membrane) or nucleic acid (cytoplasm). When
Griffith separated the heat-killed S cells into cytoplasm and cell wall/
membrane, S cytoplasm did not transform R cells into S cells. How-
ever, the S cell wall/membrane portion of cells was able to transform
R cells into S cells. When Griffith washed the cell wall/membrane por-
tion a couple of times with buffer, the S cell wall/membrane no longer
transformed R cells into S cells! Why? Griffith concluded the heritable
material for converting an R strain into an S strain was loosely attached
to the cell wall/membrane and could be washed off into the buffer.
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Because the cell wall/membrane is composed of protein and not DNA,

Griffith supported the widely held hypothesis that protein was the heri-
table material. However, he never determined what was washed off the
cell wall/membrane portion.
Griffith conducted many more experiments but he was never any
closer to showing with compelling evidence that proteins were the herita-
ble material. Nevertheless, his work was significant because he developed
an experimental system that used heritable material to transform harmless
R bacteria into lethal S bacteria. Griffith demonstrated that the “S factor”
produced more S cells as well as genetic information being stably com-
municated from parent to offspring.
Science often progresses through the dogged determination of some-
one trying to answer a vexing question. Griffith made a significant con-
tribution, but his evidence about the source of heritable material was
based on inconclusive data. Sixteen years after Griffith, another physician
by the name of Oswald Avery tried to determine the chemical composi-
tion of heritable material. Avery earned his M.D. in 1904, and 3 years
later he sought more challenging work so he became an academic scholar
at a clinic in Brooklyn, New York. He taught courses, treated patients,
and conducted research similar to the type of work conducted by medi-
cal school faculty today. Avery retired from the faculty in 1943, having
worked for 39 years. Most people his age wind down their careers, but
Avery would launch his most significant research and publish a ground-
breaking result 40 years after earning his medical degree.
Avery addressed the open question Griffith left hanging: Is protein
really the heritable material? Avery and his collaborators developed
a robust protocol to convert R cells to S cells that worked better than
Griffith’s protocol:
1. Grind up S cells in buffer to extract all soluble material (10 mL).

2. Add 50 mL ethanol to the extract. Mix and store in a refrigerator for
8 hours. A white fluffy web of stringy material will appear that looks
like a tangle of silk thread (Figure 2).
3. The next morning, centrifuge the mixture (60 mL) to pellet the
white stringy material. Pour off the ethanol, and allow the white
pellet to dry.
Figure 2 The heritable material produces S cells from R cells.

Modern photograph of transforming factor precipitate. Note the wiry
white strands in the tube.
Source: Original photo.
4. Dissolve the pelleted material in 10 mL buffer.

5. Add the sterile solution from step 4 to R cells and incubate at 37° C
for a day.
6. The next day, spread the cells from step 5 onto agar plates, and look
for the transformation from R to S colony shape. These S cells could
have been injected into mice to demonstrate lethality, but out of
concern for the animals, investigators in the 1940s had stopped
using live mice for the pneumonia test.
Avery’s team precipitated the transforming S-factor after using buffer

to remove it from the cell wall/membrane. However, they could not be
Table 1 Comparison of four preparations of transforming factor versus

purified DNA.
sample # % nitrogen, N % phosphorus, P N / P ratio
37 14.21 8.57 1.66
38B 15.93 9.09 1.75
42 15.36 9.04 1.69
44 13.40 8.45 1.58
pure DNA 15.32 9.05 1.69
Source: From Avery et al., 1944. Table I. Originally published in Journal of Experimental Medicine.
Vol:79-137-158.
certain the water-soluble DNA was pure, because proteins are also water
soluble. Chemical tests developed in the 1940s helped Avery’s team to de-
termine that the solution contained no detectable levels of protein and was
highly enriched with DNA. They analyzed the chemical composition and
compared their heritable material to a known solution of DNA (Table 1).
Jumping to conclusions is easy to do in research, because investigators
can get excited and want to move to the next interesting question. Avery
and his collaborators would have been very tempted to jump up and down
screaming, “DNA is the heritable material, not protein!” However, great
scientists are their own harshest critics and have learned to slow down and
consider all possible interpretations. How could Avery be certain that the
“S-factor” did not contain a mixture of DNA and protein? Perhaps water
soluble protein really was the transforming factor instead of DNA. The
investigators added two protein-destroying proteases, trypsin and chymo-
trypsin, to their DNA solution and discovered the DNA solution was still
able to convert R cells into S cells. These two proteases only cut between
particular amino acid pairs and do not cut every protein completely into
individual amino acids. The purified S-factor was incubated with an
RNA-destroying enzyme called RNase, and Avery was still able to trans-
formed R cells into S cells. Unlike proteases, RNase does cut RNA into
individual nucleotides. Remember that proteins are composed of amino
acids while DNA and RNA are composed of nucleotides (Figure 3).
The investigators added DNase to the “S-factor,” which eliminated the
ability of the solution to transform R cells into S cells.
Non-scientists use the term “prove” much more frequently than
scientists do because, outside of mathematics, it is impossible to prove
N C
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O O O O O
threonine glutamate alanine cysteine histidine
deoxyribose (sugar)
O
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N
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N C base (guanine)
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Figure 3 Chemical composition of amino acids and nucleotides. Above

is shown five amino acids linked together. Below are the four DNA
nucleotides linked together.
Source: Original art.
anything in science. A person can accumulate large volumes of data to

support a particular conclusion, but it is not possible to prove a biologi-
cal conclusion. To prove something, one has to formulate an explanation
against which there is not one single possible alternative explanation. In
math, it is possible to prove that opposite angles in a parallelogram are
congruent. In biology, it is impossible to prove that all cells come from
preexisting cells, or that one species gives rise to only the same species.
Compared to other sciences, biology frequently develops general rules but
adds exceptions that can be frustrating to mathematicians. However, it
is possible to disprove something, which was at the heart of Avery’s final
experiments.
Avery’s DNA solution probably did contain trace amounts of pro-

tein, which were not completely destroyed by the two proteases used by
Avery’s team. Therefore, it is impossible to prove that trace proteins were
not the genuine source of heritable material. Rather than arguing that
the DNA solution lacked any protein contamination, Avery argued that
DNase causing the loss of transformation provided compelling evidence
that DNA is the heritable material, though he could not prove it.
Despite Avery’s compelling data, some of his contemporaries were
not willing to let go of the mathematical reality that twenty amino acids
make a better genetic code than four nucleotides. Skeptics argued that
DNase released trace amounts of S-factor protein that had lost its func-
tional shape once the DNA was digested. Holding onto preconceived
ideas and misconceptions is a common human trait. The goal of science
is to use data to shape ideas—not personal bias or preferences. Neverthe-
less, all science is conducted in a human context, and Avery’s work was
regarded as significant, but not definitive. In 1944, many biologists were
not willing to concede that Avery’s team had demonstrated that DNA is
the heritable material.
Scientists are driven to answer questions, solve mysteries, and under-
stand information contained in nature. Avery did not set out to win a
Nobel Prize, but he did intend to answer one of the burning questions of
his time—is DNA the heritable material? DNA molecules are the source
of genetic information as we know now. Avery’s work was inspirational to
many and led others to consider DNA as the heritable material. As a giant
in biology and arguably one of the original molecular biologists, Avery
stood tall on the shoulders of Griffith. Avery’s work, and the end of World
War II, stimulated many others to determine the source of heritable ma-
terial. Chapter 3 presents a particularly impressive effort by one scientist
and his young technician to rule out proteins as the heritable material.
Bibliography
Anderson TF. Techniques for the preservation of three-dimensional
structure in preparing specimens for the electron microscope. Trans-
actions of the New York Academy of Sciences 13(4):130–134, 1951.
Avery OT, MacLeod CM, McCarty M. Studies on the chemical nature of

the substance inducing transformation of pneumococcal types. J Exp
Med 79(2):137–158, 1944.
Belanger AE, Clague MJ, Glass JI, et al. Pyruvate oxidase is a determinant
of Avery’s rough morphology. J Bacteriol 186(24):8164–8171, 2004.
Griffith F. The significance of pneumococcal types. J Hyg (Lond)
27(2):113–159, 1928.
CHAPTER 3
Disproving Proteins are the

Heritable Information
By the end of World War II, biologists were getting close to identifying
the heritable material. However, it is much easier to disprove an incorrect
hypothesis than it is for a community to agree on a single explanation.
It seemed probable that DNA was the molecule that carried inherited
information, but no one had provided irrefutable evidence that proteins
were not the heritable material. In 1952, a pair of biologists from Cold
Spring Harbor Laboratory on Long Island, New York, attempted to show
beyond any reasonable doubt that DNA, and not protein, was the heri-
table material. Scientists often are enamored with solving problems so
difficult that most others have failed. Others had supported Avery’s con-
clusion that DNA was the genetic information, but Alfred Hershey and
his laboratory assistant, Martha Chase, accepted the challenge to produce
overwhelming evidence. No one had performed an experiment to finally
reject the protein inheritance hypothesis. Only 2 years out of college,
Chase would take part in one of the most famous biology experiments of
the twentieth century.
Hershey and Chase set out to demonstrate a negative, that protein was
not the heritable material. In order to demonstrate a negative, essentially
the harshest skeptic must be convinced that only one plausible answer
exists and that all other answers are highly improbable. To accomplish this
very difficult task, Hershey and Chase benefited from the newly discov-
ered atomic isotopes from which they could produce radioactive DNA
and proteins. The reader may remember that many atomic isotopes (such
as, uranium and plutonium) are unstable chemical elements that decay
and emit radiation in the process. Any given isotope emits a characteris-
tic energy level of radiation. The radiation can be detected with sensitive
instruments as well as x-ray film. Hershey and Chase used radioactive iso-
topes of sulfur (written 35S, but pronounced “S 35”) and phosphorous
(32P, pronounced “P 32”). Radioactive isotopes 35S and 32P emit very dif-
ferent levels of energy, which can be distinguished from one another. Phos-
phorous was chosen for making radioactive DNA, because DNA contains
many phosphates (see Figure 3). Sulfur was chosen for making radioac-
tive proteins because, unlike DNA, proteins contain sulfur in amino acids
cysteine (see Figure 3) and methionine with each amino acid containing
one atom of sulfur. Therefore, Hershey and Chase realized that they could
produce radioactive DNA or protein and determine which isotope was
present based on the type of radiation each isotope emitted.
Hershey and Chase combined radioactive DNA and protein with a
new experimental system that became popular among geneticists after
World War II—phages which are viruses that infect bacteria. Viruses are
the ultimate parasites; they are dependent upon their hosts for their physi-
ological functions, including reproduction. Because viruses are not cells,
new phage must emerge from their larger bacterial host cells. Phage T2 was
an ideal model system, because it cannot infect humans and they repro-
duce rapidly with one parental virus yielding over 100 progeny viruses in
under an hour. T2 phage consist of a protein coat surrounding a DNA
genome. Phage proteins could be labeled, or tagged with radioactivity,
using 35S, and the viral DNA could be labeled with 32P. If the investigators
could rule out either DNA or protein as the heritable material, then the
other molecule had to be the genetic material. Hershey and Chase realized
their phage system was perfect for answering a major research question.
It would have been too difficult to chemically synthesize radioactive
DNA or protein in vitro, so like any smart biologist, they let nature do the
hard work for them. Hershey and Chase grew T2 phage inside E. coli hosts
in the presence of either radioactive amino acids or DNA nucleotides with
each experiment conducted in separate tubes. Hersey and Chase produced
radioactive viruses and their bacteria hosts (Figure 4). Because radioactiv-
ity is invisible, the investigators used a convenient way to assay whether a
population of virus contained 35S viral proteins or 32P viral DNA based on
the amount of energy emitted.
Hershey and Chase hypothesized that if DNA were the heritable ma-
terial, then the only role of the proteins was to deliver the DNA. The viral
Disproving Proteins are the Heritable Information 15
DNA
nucleotides
amino
acids
normal radioactive radioactive

phage phage phage
A
phage
ghost
turn on
blender
E. coli phage DNA
Figure 4 Hershey-Chase experimental method. A, Phage were grown

with either radioactive amino acids to label all the proteins, or with
radioactive nucleotides to label the DNA. B, In the normal phage
infection cycle, the virus injects its DNA into a much larger E. coli
cell. A blender was used to create shear force to pull the phage head
away from the infected bacterium.
Source: Original Art.
DNA would remain inside the infected bacteria, the viral protein coat
would be torn off by blending, and the bacteria would remain intact.
They used a common kitchen blender to separate the infecting virus from
its host, but they had no idea how long to pulse the blender.
Their experiments were the first time anyone had used a blender to
tear off viruses from the surface of E. coli. They allows viruses with either
32
P-labled DNA or 35S-labeled proteins to infect E. coli for 5 minutes.
They tried a variety of times but found that two minutes was sufficient to
remove most of the radioactive protein from the surface of the bacteria.
Even with zero blending, some of the 32P-labled DNA spilled outside the
bacterial cells, as did some of the 35S-labeled protein so the process was
not perfect. Real research produces imperfect data.
When only the proteins were radioactive, about 80% of the radioac-
tive protein was in the media and therefore 20% was still with the infected
cells. Conversely, 70% of the radioactive DNA was with the infected
bacteria with only 30% in the media. At this point, it was still possible
that 20% of protein associated with the cells was the heritable material
for those who preferred the protein inheritance hypothesis. Hershey and
Chase worked very hard to locate the protein not released into the media.
They determined that much of radioactive protein pieces were loosely
associated with the outside of infected E. coli. Hershey and Chase were
surprised by their own results, because just one year before the blender ex-
periment, Hershey and Chase had incorrectly published that radioactive
viral protein had been incorporated into the second generation phage.
Their reversal of conclusions is science at its best. When a better method
produces results that contradict earlier conclusions, the erroneous in-
terpretation must be modified or retracted. Hershey and Chase wanted
to demonstrate that proteins were not the heritable material. A diehard
protein hypothesis supporter could justifiably argue that the amount of
protein necessary for inheritance was the 20% associated with infected
E. coli cells. However, their next round of experiments provided more
conclusive results.
If biology was going to explain the mechanism of inheritance, it was
essential to be clear about the heritable material. Once biology could ex-
plain how offspring came to resemble their parents, biologists could study
genetic diseases and produce improved crops to feed a growing world
population. At this point in the debate, every biologist had to use evi-
dence to determine if DNA was the heritable material, 70% of which was
inside infected bacteria. When Hershey and Chase labeled phage DNA
with 32P, they detected second generation phage whose DNA also con-
tained radioactive 32P. The two meticulous investigators refined their ex-
perimental technique and produced more compelling evidence (Table 2).
Hershey and Chase wanted to finally answer the question of whether
protein or DNA was the heritable material. When confronted with data
Table 2 Location of phage protein and DNA after infection of E. coli.

sample source extracellular intracellular
35
S protein first experiment ~80% ~20%
32
P DNA first experiment ~30% ~70%
35
S protein refined experiment ~99% ~1%
32
P DNA refined experiment ~30% ~70%
Source: Data from Hershey, A. D. and Martha Chase. 1952. Originally published in Journal of
General Physiology. Vol:36-39-56.
and two possible sources of genetic information, scientists typically choose

the simpler answer, a principle known as Occam’s razor. After careful con-
sideration of both possibilities, biologists reached a consensus that DNA
is the source of genetic material, not protein. As cautious scientists, Hershey
and Chase did leave an opening for an alternative explanation—the pro-
tein responsible for heritable material might lack sulfur and thus escape
their experimental detection. Once again proof is impossible. Hershey and
Chase accepted the challenge of their day and finally convinced every-
one that DNA was the heritable material. The next wave of biochemists
wanted to determine how DNA could replicate and produce equivalent
copies of itself.
Ethical, Legal, Social Implications: DNA Ownership

Each year, biologists sequence DNA faster and cheaper to determine the
order of base pairs in chromosomes, and they can determine the exact
sequence of bases in any person’s genome. As often depicted in movies
and television, DNA sequences can be used to identify a person due to
the uniqueness of their DNA. However, DNA sequencing technology has
presented many ethical problems as well as some common misconcep-
tions, some of which come from movies and television.
First let’s dispel the most common misconceptions:
MYTH #1: DNA sequencing is done almost instantaneously.

TRUTH: It takes several hours to isolate DNA from an environmental
sample (such as a dried blood stain in clothing) and process it for
sequencing. DNA cannot be seen directly by holding a light over it.
MYTH #2: A person’s entire DNA sequence is used in criminal cases.

TRUTH: DNA legal evidence consists of very short fragments. The
pieces are so small that an analogous photo might consist of post-
age stamp-sized fragments showing an earlobe and an elbow. Iden-
tifying anyone is difficult from these two partial photos. Although
DNA sequencing is getting cheaper, we use less than 0.000001%
of a person’s genome for “DNA fingerprints.”
MYTH #3: DNA evidence is properly used to convict criminals.

TRUTH: Because we use such small portions of DNA for identifica-
tion, DNA evidence should be used to find innocence and not
guilt. Let’s return to the ear and elbow photo analogy. Imagine
comparing the photos to many different people. Two people might
have ears and elbows that look alike, but only one committed the
crime. However, if someone has pierced ears or scars on their el-
bows is photographed, then one can be certain the person with
altered ears is not the criminal. Likewise, it is appropriate to say
that a suspect is not the criminal if the DNA evidence differs from
the suspect. It is inappropriate to say that two matching samples of
DNA prove the evidence came from the suspect.
MYTH #4: DNA evidence is infallible.

TRUTH: Producing DNA evidence is complex and performed by
humans, therefore, mistakes happen. Perhaps the biggest mistakes
are made in handling the evidence. What if a person’s dead skin
cells fell onto the sample? What if someone wanted to frame an
enemy and put that person’s DNA at the scene of a crime? DNA
evidence is no more infallible than the people doing the work.
Today, there are no national standards required of labs that analyze
DNA samples. Project Innocence website provides a lot more in-
formation about this topic.
DNA sequence information has many other applications beyond

forensics. In clinical settings, DNA can be used to diagnose some ge-
netic diseases directly or to indicate a greater than average potential to
develop some diseases. Today, it is possible to get home genetic kits that
make claims of determining a person’s risk for certain diseases. But hav-
ing access to this information presents new challenges. What if a person
wants to be tested for Huntington disease, a genetic disease that is fatal
100% of the time, and they find out they have it? Who should they tell?
Everyone in this person’s family would know that one of their parents
has Huntington disease too. What if their employer found out, and they
devised a plan to fire the person who had the DNA test to save money on
insurance costs? Once a person learns his or her ultimate fate, would the
person behave differently? Do people really want to know their futures if
they cannot alter it? And what if the genetic test was wrong and the per-
son did not have what the results indicated? Just as patients have gone to
hospitals and mistakenly had a limb amputated, DNA tests can produce
erroneous results.
Another problem of improved DNA sequencing is that we leave our
genomes everywhere we go. When a person sheds hair, sneezes, coughs,
licks stamps, or skins a knee, the person is leaving hundreds or thousands
of copies of his or her full genome for someone else to collect. If they
shed a hair and let it fall on the ground, someone else could pick it up
and use it without their permission. A person’s heritable material is avail-
able to anyone with access to DNA sequencing technology. Companies
will sequence any sample of DNA with no questions asked. In 2015, it
is possible to can get 1000 bases sequenced for $2.00, and in the future,
this probably will cost $0.02. Improved DNA sequencing means that we
might all be vulnerable to DNA identity theft.
Typically, DNA is faithfully replicated from one generation to the
next. However, over time, slight changes or mutations accumulate so
that we have enough variation to be able to tell two people apart, even
“identical” twins. There are over 7 billion people alive today, and nearly
all of them have a unique sequence. The experiments conducted by the
pioneers of genetic research had no idea that their work would lead to the
potential to diagnose diseases, exonerate falsely accused suspects, or facili-
tate DNA identity theft. They were simply trying to answer questions that
had eluded others and were critical to our understanding of life.
As is often the case, knowledge is not good or bad by itself, but the
application of that knowledge can be as diverse as the human population.
To learn about a terrible use of genetics that occurred in the United States
during the twentieth century, visit the Cold Spring Harbor Laboratory
Eugenics Archive website. It is important to understand and be aware of
abuses in science so that these harmful practices cannot be repeated later
under a new name or with noble-sounding slogans.
Bibliography
Hershey AD, Chase M. Independent functions of viral protein and nu-
cleic acid in growth of bacteriophage. J Gen Physiol 36(1):39–56,
1952.
www.Ebook777.com
CHAPTER 4
DNA Structure Determines

Its Function
In the 1950s, biologists wanted to know the structure of DNA because,

as a widely accepted rule in biology states, structure determines func-
tion. If one can understand how an object is built, then it should be pos-
sible to deduce how the object works. No doubt everyone has examined
simple machines, such as a pencil sharpener or can opener, and deduced
the mechanism of its action. Understanding how DNA works, through
knowledge of its structure, could allow biologists to determine the cause
of genetic diseases and possibly find cures. With this underlying reduc-
tionist principle in mind, some of the best chemists, biochemists, and
physicists competed with one another to be the first to accurately describe
DNA’s structure. Many had tried unsuccessfully, including Nobel laure-
ate Linus Pauling, who had proposed in 1953 an incorrect structure that
consisted of DNA coiled in three strands with sugars and phosphates in
the center and nitrogenous bases sticking outward. Interestingly, James
Watson and Francis Crick had reached a similar, incorrect conclusion,
but they later rejected the sugar-inside model while searching for a better
model of DNA’s structure.
The Structure of DNA

The folklore around the discovery of DNA’s structure is convoluted and
full of allegations of deceit and/or hostility, but this chapter focuses on the
science rather than the sociology. The major problem of the day was the
lack of sufficient data to determine definitively the overall structure. Pre-
vious research had revealed that DNA was composed of carbon, nitrogen,
phosphorous, oxygen, and hydrogen; but chemical composition does
A B
O– O
P 3 phosphates H
–
O O O N
N
P C H
C
–
O O O adenine
N C
P C N
– 5` H
O O C O
N C
C 4` C
H 1`
3` C C
2`
O H
H
deoxyribose
C D
Figure 5 Nucleotide structures. Atoms are located at the bent corners,

and covalent bonds between them are colored rods. A, Used for energy
and RNA synthesis, adenine triphosphate (ATP) looking at the face
of the pentagonal ribose in the middle, three phosphates extending
to the left, and the base (adenine) seen on edge. B, Used for DNA
synthesis, deoxyribose adenine triphosphate (dATP) lacks one oxygen
on the bottom right corner of the pentagon ribose. C, Rotated view of
dATP with the adenine base displayed fully on the right side. D, Line
diagram of dATP.
not determine DNA’s overall shape (Figure 5). They also knew that the
basic building blocks consisted of nucleotides, which have three parts: a
phosphate group, a nitrogen-containing base, and a 5-carbon sugar called
deoxyribose. There are four bases in DNA: guanine, cytosine, adenine,
and thymine (abbreviated as G, C, A, and T; see Figure 3). Nucleotide
monomers are composed of three phosphates, but when they are con-
nected together in DNA polymers, there is one phosphate for every base,
which means two of the phosphates are removed when incorporated into
a polymer of DNA. In Figure 5, one can see the difference between nucle-
otides in DNA and RNA. DNA nucleotides contain deoxyribose, which
lacks one oxygen atom compared to ribose in RNA. DNA nucleotides are
abbreviated dGTP, dCTP, dATP, and dTTP, or dNTP collectively. RNA
DNA Structure Determines Its Function 23
nucleotide abbreviations lack the “d” because its ribose retains the oxygen
absent in DNA. Compare the structures of ATP and dATP in Figures 5A
and 5B. Each carbon vertex in a pentagon-shaped ribose is numbered
clockwise as 1′, 2′, 3′, 4′, and the 5′ which is outside the pentagon; the 1′
carbon supports the base (Figure 5D).
As the various research teams raced to be first to discover the structure
of DNA, a collection of three British labs working in close proximity
all arrived at the same idea at about the same time. Whenever a person
works in a group that produces a good idea, it is often difficult to remem-
ber who had the idea first. Because everyone knew that determining the
structure of DNA would garner a Nobel Prize, the three groups needed to
coordinate how to publish their papers. The compromise was to have one
journal publish all three papers in a row. On April 25, 1953, the journal
Nature published papers by James Watson and Francis Crick on page 737,
Maurice Wilkins, Alex Stokes, and Herbert Wilson on page 738, and
Rosalind Franklin and Raymond Gosling on page 740. These three pub-
lications were so significant that April 25th is now designated as National
DNA Day in the United States.
Watson and Crick’s paper began by criticizing Linus Pauling’s triple-
stranded model, arguing that three strands of negatively charged phos-
phates would repel each other and cause the molecule to fall apart, just as
negative ends of magnets repel each other. The two authors enumerated
data that others had already published, just as one would organize all the
pieces of a jigsaw puzzle before assembling it. Research had shown that
there was more than one helix, probably two, and they spiraled in the same
direction. Each nucleotide was attached to the adjacent one, with the 3′
carbon of one nucleotide attached to the 5′ carbon of the next nucleotide,
with a phosphate in between the two nucleotides (see Figures 3 and 5).
The two investigators also knew from prior research that the two strands
were antiparallel, meaning they ran in opposite directions with the 5′ end
of one strand pointing one way and the 5′ end of the other strand point-
ing the opposite way. “We wish to put forward a radically different struc-
ture of [DNA]. The structure has two helical chains, each coiled round
the same axis.” Unlike Pauling, Watson and Crick placed the bases in
the center, with 10 bases every 34 angstroms (abbreviated Å; 1 × 10210
meters) and a uniform diameter of the entire double helix equal to 20 Å.
category analogy chemical representation common in

H O
hydrogen + – (N or O) and H
bond N H O C
H O
+ –
ionic bases (+) and
N H O C
bond acids (–)
H
H
single any elements, often
covalent N C C–C, C–O,
bond double
O C–N, C–H
Figure 6 Three types of chemical bonds. Hydrogen bonds are

the weakest and form between two partially charged, δ, atoms
(frequently nitrogen or oxygen) with hydrogen. Ionic bonds are
intermediate in strength and form between two fully charged atoms
of opposite polarity, frequently between an acid and a base. Covalent
bonds are the strongest, can be single or double bonds, and are
formed by sharing electrons.
“The novel feature of the structure is the manner in which the two chains
are held together by the purine and pyrimidine bases.” Purines (G and A)
contain two rings; pyrimidines (C and T) contain one ring. Watson and
Crick described the bases as lying in a plane perpendicular to the long
axis of DNA. They correctly understood why DNA would have equal
numbers of A’s and T’s and equal numbers of G’s and C’s, because they
were base-paired, or bound, to each other. The base pairs hold on to each
other through weak hydrogen bonds (H-bonds; Figure 6). Hydrogen
bonds are very weak and can only span very short distances.
To help them see their proposed molecular structure, Watson and
Crick built a metal version of DNA that was taller than the two men.
Being able to walk around a physical structure, touch it, and see how
the atoms were spaced, enabled them to recognize that RNA with its
extra oxygen would not form the same structure of a long double helix.
Even after building the physical model, the pair wrote an interesting dis-
claimer in their April publication. They declared that all the previously
published images of DNA taken with x-rays are insufficient to support
their proposed model and that additional data are needed. “Some of these
data are given in the following communications,” meaning the two papers
following their paper. “We were not aware of the details of the results
presented [in the next two papers] when we designed our structure which
rests mainly, though not entirely, on published data . . . .” That is a shock-
ing statement. Watson and Crick used unpublished data to reach their
conclusions, and yet they did not publish the data they used. Their dis-
claimer implies they used the data of others, but whose? In their closing
they state, “We have also been stimulated by a knowledge of the general
nature of the unpublished experimental results and ideas of Wilkins and
Franklin and their co-workers.” Now we know whose data they used, even
though they did not publish as a single and unified paper.
Watson and Crick are famous for determining DNA’s structure, and
professional biologists know the unusual circumstances surrounding their
use of someone else’s unpublished data. Another equally famous aspect of
the Watson and Crick two-page paper is perhaps the most understated
sentence in any Nobel Prize winning research. “It has not escaped our no-
tice that the specific base pairing we have postulated immediately suggests
a possible copying mechanism for the genetic material.” In other words,
because of the structure, they understood the function of DNA as genetic
information to be replicated and passed on to the next generation. When
someone knows how a simple machine is constructed, he or she knows
how it works: form follows function.
In the paper by Wilkins et al., the authors included a photo of a DNA
x-ray diffraction pattern. The paper described how this image led experts
in the field to deduce the Watson and Crick structure on the previous page
in the same journal. Beginning on the same page where the Wilkins paper
ended, Franklin and Gosling explained their understanding of DNA and
they published a much better DNA x-ray diffraction pattern. As usual,
the two authors began by restating what had been published previously,
including the 1949 PhD thesis of a graduate student by the name of Fur-
berg. From the Furberg data, several other scientists had predicted that
DNA was helical in nature. Franklin and Gosling reiterated what Watson
and Crick published just a few pages earlier describing the uniform 20 Å
diameter and 3.4 Å separation between bases. Franklin and Gosling
concurred that the phosphates were on the outside with bases radiating
inwards. They noted that DNA is composed of two chains, not three,
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and the two chains are not equally spaced in the long axis of the polymer.
They concluded, “Thus our general ideas are not inconsistent with the
model proposed by Watson and Crick in the preceding communication.”
In short, they said ditto. If the reader wants to understand how x-rays
were used to deduce the structure of DNA, use an online tutorial to learn
how a pattern of dark spots revealed the DNA structure.
About one month after the April 25 three-way publication describing
the structure of DNA, Watson and Crick published another paper whose
title overcompensated for their previous understatement, “Genetical Im-
plications of the Structure of Deoxyribonucleic Acid.” Oddly, they began
by taking a small step backward by stating, “Many lines of evidence indi-
cated that [DNA] is the carrier of a part (if not all) of the genetic specificity
of the chromosome. . . .” They attempted to leave some wiggle room, sug-
gesting that perhaps protein could carry some heritable information de-
spite all of the compelling evidence from Hershey and Chase. Watson and
Crick rehashed their previous month’s paper before offering greater detail.
They stated that if they knew the sequence of bases on one strand, they
could deduce the sequence on the other strand because of the required
base pairings via H-bonds. As the weakest bond, H-bonds cannot span
distances greater than 2 Å. “Thus one chain is, as it were, the complement
of the other . . . which suggests how the deoxyribonucleic acid molecule
might duplicate itself.” They continue by stating that DNA is not a tem-
plate but really a “pair of templates” with each strand serving as the foun-
dation upon which the other strand could be polymerized from nucleotide
monomers (dNTPs). They were uncertain if an enzyme catalyzed the rep-
lication of DNA or whether DNA was able to spontaneously self-assemble
the monomers into a polymer. Based on the distances in their model, they
noted that a single polymer of protein could fit into the major groove
of DNA, thus providing a way for the protein inheritance hypothesis to
survive a bit longer. Because Watson and Crick did not experimentally test
their DNA structure, they stated their scheme was “speculative,” though
subsequent research showed they were mostly correct, as we will see.
Several books have been written about the very lively race to deter-
mine the structure of DNA. In the end, Watson, Crick, and Wilkins
earned the Nobel Prize, but Franklin did not. Many have claimed that
Franklin was inappropriately denied the award because she was a woman,
but Nobel laureates must be alive at the time of the award and unfor-
tunately, Franklin died from cancer when she was young. Nevertheless,
while she was alive, Franklin may have been denied the accolades that she
deserved because of her gender. Her x-ray image of DNA is clearly the
better one, and yet her paper appeared third in a series of three. Despite
their legendary discovery, Watson and Crick mistakenly drew the G and
C base pair with only two H-bonds instead of three, and the two strands
of DNA as parallel instead of antiparallel. Although there is sufficient
space for a protein to bind in the major groove, Hershey and Chase had
already demonstrated that protein was not the heritable material.
The race for determining the structure of DNA was over, and its struc-
tural beauty was satisfying in many ways. However, all scientific discover-
ies lead to more questions. This time, the looming question was: How is
DNA information replicated (that is, how does DNA polymerase polym-
erize dNTPs into a polymer that forms chromosomes)? It was presumed
that the two strands separated and each became a template for its comple-
mentary strand. For instance, every G on a template strand would recruit
a C for the new strand. The next major question in biology centered on
how one double-stranded DNA molecule was replicated into two equiva-
lent double-stranded DNA molecules.
DNA Replication
In the mid-1950s, three hypothetical models emerged to explain how the
double helix of DNA could be replicated to retain the original informa-
tion of the parental cell and still provide equivalent DNA to be passed
on to the next generation (Figure 7). One hypothesis stated that each old
strand was the template for a complementary new strand so that each of
the two new DNA double helices would be composed of one old strand
and one new strand (Figure 7A). Another hypothesis proposed that the
original DNA double helix would be left intact, and the new DNA mol-
ecule would be composed of two completely new strands (Figure 7B).
The final possibility was that the old strands were fragmented into smaller
pieces that would serve as templates so that each of the resulting two
double-stranded DNA molecules would be a mosaic of old and new seg-
ments (Figure 7C). Biologists were stymied and could not think of an
A B
Figure 7 Three possible models for DNA replication with one piece
of double-stranded DNA (dsDNA) in the center (two dark colors).
A, Each original strand is a template for the other strand, and the
new dsDNA molecules have one old strand (dark) and one new strand
(pale). B, The original molecule remains intact (dark), and the new
double helix is composed of two new (pale) strands of DNA. C, Each
strand of the old is a template for the new, but all four strands of
DNA are a mosaic of old and new DNA.
Source: Original art modified from Meselson, Matthew and Franklin W. Stahl. 1958.
experimental approach to distinguish these three models. Into this void

stepped a chemist and a biochemist from Caltech who devised what has
been called “the most beautiful experiment in biology.”
Matthew Meselson and Franklin Stahl wanted to determine which
model in Figure 7 was correct. They made two critical decisions in their
choice of experimental system: study DNA replication in the bacterium
E. coli, and use stable isotopes of nitrogen to label DNA. By contrast,
remember that Hershey and Chase used radioactive isotopes. Meselson
and Stahl used non-radioactive isotopes—heavier 15N than the more
abundant, lighter form of nitrogen 14N. Their goal was to alter the ratio
of heavy and light nitrogen in growth media as E. coli replicated and
thus force the cells to incorporate different isotopes into the nitrogen-rich
bases of G, C, A, and T. They anticipated that the heavy and light DNA
could be separated by centrifugation and that the relative amounts of
heavy and light DNA would support only one of the models in Figure 7.
First, Meselson and Stahl needed to develop a new method of ana-
lysing DNA from cells because DNA density would be crucial in their
experiments. DNA isolated from E. coli was centrifuged in a salt solution
for different amounts of time and then photographed using ultraviolet
(UV) light to expose the film. DNA absorbs UV light, and dark portions
on the photograph indicated the presence of DNA. The opposing forces
of sedimentation and diffusion in the salt established a concentration gra-
dient along the length of the centrifuge tube with the highest density
at the bottom of the tube. After 36 hours of centrifugation, the DNA
stopped moving because the DNA’s density matched the density of the
salt at a particular level in the tube. A photograph of DNA appeared as a
single dark band in the half way up the centrifuge tube.
Next, Meselson and Stahl needed to test their new method to deter-
mine if it was sensitive enough to detect whether DNA labeled with light
nitrogen (14N) would have a different density compared to DNA labeled
with heavy nitrogen (15N). A mixture with approximately equal amounts of
light and heavy DNA was centrifuged and photographed. The photograph
showed two distinct dark bands the width of the tube and separated from
each other by about 1 centimeter. The amount of DNA in each band could
be quantified by the intensity of the blackness in the photograph.
It is widely known that a single neutron is too small a mass to measure
easily. Therefore, Meselson and Stahl could never measure the difference
between a single nucleotide of heavy or light DNA. The only way that
they could detect the difference in mass between heavy and light DNA
was to use very long strands of DNA so that each molecule would have
millions of either lighter 14N or heavier 15N. The accumulated differences
would permit the two different types of bacterial DNA to have differ-
ent masses but the same volume, which means they would have different
densities. At the start of the experiment, the DNA was layered on top of
the salt gradient with the lowest density of salt on top. Spinning the two
types of DNA in a single density gradient of salt would cause the heavy
DNA to migrate further into the salt gradient than the lighter DNA and
two bands distinct bands were visible.
Because Meselson and Stahl were not sure about the exact timing of
DNA replication in E. coli, they started two experiments in parallel. They
grew two separate flasks of cells in heavy 15N media for 15 hours, peri-
odically counting the cells to know how many were in each flask. At the
end of the 15-hour period, essentially all the DNA in the cells was heavy.
At time zero, they changed the media and started growing the cells in
light 14N so that the DNA would begin to accumulate bases made of the
lighter element while the investigators continued to monitor cell division.
Once the cells had been moved to the 14N media, they isolated DNA
from aliquots of the growing cells over the next 8 hours. At each time
point, they applied the DNA to the salt gradient, spun the samples until
they reached equilibrium, and photographed the DNA to document how
far the DNA migrated (Figure 8).
Figure 8 Centrifugation results after DNA replication. DNA was

isolated after cells were shifted from heavy to light media and spun in
the salt gradient to determine DNA density. Mixed DNA (0 and 2,
0 and 4) help distinguish relative densities of critical bands. Dotted
lines were added to use with a straight edge to distinguish slight shifts
in band positions.
Source: Modified from Meselson, Matthew and Franklin W. Stahl. 1958. Figure 4.
www.Ebook777.com
In Figure 8, it can be seen that at time zero, all the DNA contained
heavy nitrogen (15N). After one generation, all the DNA was in a sin-
gle band of intermediate density. This result eliminated the hypothesis
shown in Figure 7B which predicted two bands of 15N and 14N with equal
amounts of both. After two generations, the DNA was in two bands, inter-
mediate and only 14N. This result eliminated the hypothesis in Figure 7C
which predicted a single band of 75% 14N and 25% 15N. Generation
four shows that the intermediate DNA is always around, but it becomes
a smaller percentage of the total DNA as more and more is labeled with
light nitrogen 14N. By combining DNA from 0 and 2 generations, as
well as 0 and 4 generations, Meselson and Stah confirmed that the bands
had shifted in density and they had not made a mistake about the bands’
densities when looking at only one generation at a time. Therefore, the
hypothesis in Figure 7A, semiconservative replication, was shown to be
the correct mechanism for DNA replication.
Meselson and Stahl were heroes in the biological research community
because they had demonstrated beyond any reasonable doubt that DNA
uses semiconservative replication. Understanding how DNA replicates al-
lows biologists to determine the molecular origins of genetic diseases and
conduct research on individual DNA molecules in the lab. Meselson and
Stahl’s experimental design has been honored for years, in part because
no matter which of the three replication models was correct, their experi-
ment would have determined the correct one. Their work tackled one of
the most difficult questions of their time, but that does not mean that all
of the big questions had been answered. Does DNA replication make any
errors? If so, can these errors be fixed? How does DNA polymerase get
started? How is the molecular information in DNA converted into a pro-
tein? What turns genes on and off? Is the DNA sequence of bases the only
genetic code? In short, good research leads to more research questions.
DNA is the heritable material and its double-stranded structure facili-
tates each strand becoming a template for the production of a new strand.
So far, this book has presented the critical experimental data that led to
the conclusion that DNA is a molecular storage device for genetic infor-
mation. Most people think that DNA replication produces two identical
copies of DNA, which is not a true statement. DNA replication produces
two equivalent, but not necessarily identical, copies of DNA. In Chapter 5,
it will be shown that the information content in DNA is more compli-

cated than merely the sequences of nucleotides.
Bibliography
Franklin RE, Gosling RG. Molecular configuration in sodium thymo-
nucleate. Nature 171(4356):740–741, 1953.
Holmes FL. Meselson, Stahl, and the replication of DNA: a history of
“the most beautiful experiment in biology.” New Haven, 2001, Yale
University Press.
Meselson M, Stahl FW. The replication of DNA in Escherichia coli. Proc
Natl Acad Sci 44(7):671–682, 1958.
Watson JD, Crick FH. Molecular structure of nucleic acids: a structure
for deoxyribose nucleic acid. Nature 171(4356):737, 1953a.
Watson JD, Crick FH. Genetical implications of the structure of deoxy-
ribonucleic acid. Nature 171(4361):964–967, 1953b.
Wilkins MH, Stokes AR, Wilson HR. Molecular structure of deoxypen-
tose nucleic acids. Nature 171(4356):738–740, 1953.
CHAPTER 5
Not all DNA Information is

Linear in Nature
It may be a surprise to learn that the information content of DNA

can be modified chemically without altering its sequence of base pairs,
similar to capitalizing some letters without changing the words: tHE
LoveLy Oven. If one reads all the letters equally, it would be possible to
form an opinion about a kitchen appliance. However, if one reads only
the upper case letters, a salutation is revealed. To read “HELLO,” each
letter’s case matters, and in DNA, chemical modification of nucleotides
matters. For example, if chemical modifications can alter the informa-
tion carried by DNA, then perhaps modified DNA could help explain
some illnesses caused by environmental factors. Once biologists realized
there was more to DNA that just the order of base pairs, investigators
wanted to understand the full extent of the modifications and their
consequences.
A summary paper in 1975 described biologists’ simplistic understand-
ing of DNA modification and its effects on gene activity. The paper stated
that the appearance of an organism, its phenotype, depends solely on
the information in its DNA, its genotype. “Since the ultimate control of
development resides in the genetic material, the actual program must be
written in base sequences in the DNA.” The authors noted that bacteria
chemically modify some of their adenine (A) bases by adding a methyl
group (CH3) to the number 6 nitrogen (Figure 9C). Methylated DNA
cannot be destroyed by bacterial enzymes that cut, or restrict, DNA into
smaller pieces of unmethylated viral DNA and thus killing the invading
virus. The combination of restriction enzymes and methyl-adenine pro-
vided bacteria with a simple immune system that attacks viral DNA not
protected by methylation. The authors of the 1975 summary did note
adenine cytosine carbon

oxygen
A B
nitrogen
hydrogen
methyladenine methylcytosine
C D
Figure 9 Methylated DNA bases. (A) Adenine and cytosine (B) and
their methlyated variations (C and D). Methylcytosine is abbreviated
m5C because the methyl group is attached to carbon number 5 if
counting is started at the bottom of the ring and move counterclockwise
around the ring.
that eukaryotes (such as, animals and plants) methylate some of their
cytosines on carbon number 5 (abbreviated m5C; Figure 9D) but they
did not know why. They concluded by saying, “We are aware that no
direct evidence exists for specific modification enzymes in eukaryotes,
let alone that such enzymes might exercise control of gene activities.”
Because so little was understood about the control of eukaryotic gene
activity in the 1970s, the authors argued that biologists should look for
DNA methylation enzymes in eukaryotes.
Not all DNA Information is Linear in Nature 35
TL TL
components
of sample
mixed
sample
solvent A solvent A
A B
TL Rotate 90°
TL
TL
solvent B solvent B
C D
Figure 10 Two dimensional thin layer chromatography (TLC)

technique. A, Complex mixture of a sample is pipetted on the line and
then the bottom edge is dipped in solvent A. B, Sample components
migrate at different rates depending on their chemical structures.
C, The sheet is rotated and dipped into solvent B where (D) the
components again migrate according to their chemical properties. TL
marks the top left corner in A to help with orientation in C and D.
Over the next few years, investigators learned a lot about methyl-
ated cytosine in eukaryotes. As the methods for DNA sequencing became
commonplace, a pattern emerged—DNA segments rich in C and G bases
were frequently methylated, and these DNA segments often controlled
when a gene was turned on or off. To detect when cytosine had been
methylated, biochemists Tally Naveh-Many and Howard Cedar adapted
a common technique used by chemists. For many years, chemists had
used thin layer chromatography (TLC) to separate molecules with dif-
ferent chemical structures (Figure 10). The biologists added radioactive
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phosphorous to every nucleotide in the eukaryotic DNA and then sepa-

rated the complex mixture of nucleotides using TLC. The investigators
were able to separate five different bases (G, C, A, T, and m5C). With
TLC, the investigators compared the methylation status of C- and G-rich
segments in previously purified DNA versus DNA freshly isolated from
active nuclei.
The data from this first experiment, as well as many other experi-
ments, led to a generalization—hypermethylated (over-methylated)
DNA is often silent and hypomethylated (under-methylated) DNA is
often active. The trend of silenced genes being methylated led to a testable
hypothesis. Perhaps epigenetic modifications of DNA could alter gene
activity and lead to diseases. To test this hypothesis, a team from the Uni-
versity of Illinois Medical School wondered if they could alter epigenetic
methylation of genes to turn on a gene of interest. Although genome-
wide alteration of methylated DNA might affect many genes, new bio-
medical treatments often start with broad, non-specific experiments that
can be refined later to minimize side effects if the first experiments are en-
couraging. The investigators hoped to use epigenetic DNA methylation
to determine if people with sickle cell disease could be helped without
changing the DNA sequence of their genomes.
Sickle cell disease is caused by a gene that encodes for a malfunction-
ing protein. The aberrant protein is encoded by a hemoglobin gene. All
primates, including humans, have several hemoglobin genes that encode
the proteins responsible for carrying oxygen in our red blood cells. Sickle
cell disease hemoglobin causes red blood cells to become deformed and
sometimes resemble a sickle, or a crescent moon. The abnormally-shaped
red blood cells clog the smallest capillaries, leading to many circulatory
problems and tissue damage. The molecular information that causes
sickle cell is in the DNA sequence of every cell in the patient’s body. The
investigators wanted to block the disease symptoms by turning on an
alternative hemoglobin gene that was not mutated but normally silent in
adults. Their idea was to use epigenetic manipulation of a methylated he-
moglobin gene and replace the faulty hemoglobin protein with a properly
functioning hemoglobin.
When a person is born, his or her red blood cells were filled with
a fetal form of hemoglobin encoded by a different gene than adult
Not all DNA Information is Linear in Nature 37
hemoglobin. The investigators wondered whether it would be possible

to chemically manipulate the methylation of hemoglobin genes in sickle
cell patients to activate the normally silent fetal hemoglobin gene. Other
investigators had already discovered that C- and G-rich DNA segments
adjacent to the fetal hemoglobin gene became hypermethylated 6 months
after birth, perhaps explaining how this gene could be active during fetal
development and infancy but silenced after 6 months of age. The clinical
investigators chose a drug called 5-azaC that was already shown to pre-
vent formation of m5C. The compound 5-azaC is a modified version of
the base cytosine, but it cannot be methylated at the number 5 carbon.
Because conducting preliminary research on humans would be unethi-
cal, the investigators used a monkey model for sickle cell for their first
experiments. They injected monkeys with two different dosages of 5-azaC
over a period of a couple months, and they measured the change in fetal
hemoglobin in the monkeys’ blood. The increased fetal hemoglobin indi-
cated that in monkeys, and probably humans, the fetal hemoglobin gene
activity could be increased by reducing epigenetic methylation of DNA.
Therefore, they had the first evidence that epigenetic alterations could be
used to treat diseases.
The normally silent fetal hemoglobin gene was activated when epi-
genetic methylation was blocked. The higher dose of 5-azaC did not in-
crease the maximum level of fetal hemoglobin, but the duration of the
elevated fetal hemoglobin was prolonged. Before trying this treatment on
humans, a scientist should answer some critical questions. Is it possible to
alter the methylation status of DNA in only a subset of cells, such as the
bone marrow for sickle cell disease patients? Could only a subset of genes
be targeted for epigenetic manipulation? As it turns out, 5-azaC produced
too many negative side effects to be clinically useful, but the research was
successful in that it demonstrated for the first time a phenotype change
caused by intentional epigenetic manipulation of DNA. When 5-azaC
is given to an individual, the methyltransferase enyme cannot add a
methyl to cytosine to any DNA in the entire body.
Despite the negative off-target clinical consequences of 5-azaC, the
data revealed that DNA in chromosomes carries more information than
just the sequence of bases. The epigenetic status of DNA carries infor-
mation that can alter an individual’s phenotype without changing its
genotype. The extra layer of molecular epigenetic information demon-

strates that there is additional biological information in the non-heritable
chemical modifications of DNA. Methylation of DNA can produce vari-
ation in phenotype but the methylation status is not permanent. In short,
our chromosomes carry complex layers of information beyond the four
nucleotides of G, C, A and T. Understanding epigenetic regulation of
DNA (the epigenome) is an area of active research today, largely because
we understand it so poorly. Biological information at the molecular level
must include both the genome and epigenome.
Bibliography
DeSimone J, Heller P, Hall L, et al. 5-Azacytidine stimulates fetal hemo-
globin synthesis in anemic baboons. Proc Natl Acad Sci 79(14):
4428–4431, 1982.
Gaudet F, Rideout WM 3rd, Meissner A, et al. Dnmt1 expression in pre-
and postimplantation embryogenesis and the maintenance of IAP
silencing. Mol Cell Biol 24(4):1640–1648, 2004.
Greunbaum Y, Szyf M, Cedar H, et al. Methylation of replicating and post-
replicated mouse L-cell DNA. Proc Natl Acad Sci 80(16):4919–4921,
1983.
Holliday R, Pugh JE. DNA modification mechanisms and gene activity
during development. Science 187(4173):226–232, 1975.
Jones PA, Takai D. The role of DNA methylation in mammalian epi-
genetics. Science 293(5532):1068–1070, 2001.
Naveh-Many T, Cedar H. Active gene sequences are undermethylated.
Proc Natl Acad Sci 78(7):4246–4250, 1981.
Wolffe AP, Matzke MA. Epigenetics: regulation through repression. Sci-
ence 286(5439):481–486, 1999.
Conclusion
In this book, the reader had access to data from classic experiments that
demonstrated DNA is the heritable material. Species reproduce them-
selves by providing equivalent DNA information to their offspring. The
double-stranded structure of DNA facilitates the mechanism of replica-
tion by copying each strand in a semiconservative manner so that each
cell retains its own genetic information while also supplying the next
generation with very similar information. The order of nucleotides in a
genome is critical information for each cell as is the epigenome, such as a
base’s methylation status. If either of these types of molecular information
is abnormal, the cell could die or behave differently.
The reader also has experienced how science makes progress by add-
ing to the work of others. The communication of scientific discoveries is
another way that information connects many aspects of biology. Standing
on the shoulders of giants and looking into the future for the next area of
active research is where scientists want to be in order to make new discov-
eries about the nature of biological information.
Glossary
14
N. normal nitrogen atom.
15
N. heavy nitrogen atom contains one extra neutron.
32
P. radioactive phosphorous isotope, emits strong energy level.
35
S. radioactive sulfur isotope, emits weak energy level.
angstroms. one angstrom is 1 × 10210 meters, a unit of measure ten times smaller
than a nanometer.
covalent bonds. chemical connection between two atoms held together by shared
electron pair.
deoxyribose. five carbon sugar that comprises part of a DNA nucleotide.
DNase. DNase is an enzyme that breaks DNA into individual nucleotides.
dNTP. any one of the four DNA nucleotides (dGTP, dCTP, dATP, dTTP).
emergent property. an emergent property is an unexpected consequence appar-
ent only when examining combined systems.
epigenetic. epigenetic changes to DNA are chemical modification that do not
change the sequence of DNA nucleotides.
extracellular. area outside cells.
hydrogen bonds. hydrogen bonds are weak attractions of a partially positively-
charged hydrogen atom and a weakly negative atom, such as nitrogen or oxygen
hypermethylated. DNA containing more methylated cytosine bases than is
typical.
hypomethylated. DNA containing fewer methylated cytosine bases than is
typical.
in vitro. in vitro literally means “in glass”, but, in general, it refers to experiments
performed outside live cells.
intracellular. area inside cells.
ionic bonds. chemical connection between two atoms held together by atoms of
opposite charge.
methylated DNA. DNA with methyl group (CH3) covalently attached to a
nucleotide.
methylcytosine. DNA with methyl group (CH3) covalently attached to cytosine
base of nucleotides.
methyltransferase. methyltransferases are protein enzymes that covalently attach
a CH3 methyl group onto the base of a nucleotide.
nitrogenous bases. ringed and planar organic molecules composed of purines or
pyrimidines.
42 GLOSSARY
non-radioactive isotopes. variants of atoms that do not emit radioactivity.

nucleotides. nucleotides contain a form of ribose sugar, a nitrogenous base, and
one to three phosphates.
proteases. a protease is an enzyme that destroys proteins by breaking them into
smaller pieces.
prove. demonstrating a fact without any possible alternative interpretation; proof
only possible in mathematics.
purines. double-ringed nitrogenous bases in DNA and RNA; adenine and
guanine.
pyrimidines. single-ringed nitrogenous bases in DNA and RNA; cytosine, thy-
mine or uracil in RNA.
R colonies. bacterial colonies that appeared rough (R) and were not lethal to
mice.
RNase. RNase digests RNA into individual nucleotides.
S colonies. bacterial colonies that appeared smooth (S) and were lethal to mice.
S factor. unknown material that converted R colonies into S colonies; first char-
acterization of heritable material.
semiconservative replication. semiconservative replication is the process by
which each old strand of DNA is used as the template for a new strand.
thin layer chromatography. thin layer chromatography is a method that separates
chemicals based on their ability to move on a powdery surface.
Index
Aberrant protein, 36 dNTP, 22, 26
Adenine, 22, 34 dTTP, 22
Amino acids, chemical composition
of, 8–9 Emergent property, 2
Angstroms, 23 Epigenetic modifications, of DNA, 36
Avery, Oswald, 6–10 Escherichia coli, 14–15
DNA replication in, 28–32
Bacteria, strains of, 3 location of phage protein and DNA
Biological information, definition after infection of, 17
of, 1–2
5-azaC, 37
Carbohydrates, 5 Franklin, Rosalind, 25
Cell wall/membrane, 5–6
Chase, Martha, 13–17 Gosling, Raymond, 25
Chemical bonds, types of, 24 Griffith, Fred, 3–6
Cold Spring Harbor Laboratory, 13 Guanine, 22
Covalent bonds, 24
Crick, Francis, 23–27 Heritable material
Cytoplasm, 5 chemical composition of, 6
Cytosine, 22, 34 for converting an R strain into S
strain, 5–7
DATP, 22 disproving protein as, 13–20
dCTP, 22 proving DNA as, 8–10
Deoxyribose, 22 search for, 3–10
dGTP, 22 Hershey, Alfred, 13–17
DNA Hundredth Monkey, The, 1
analysis of, 29 Huntington disease, 19
as heritable material, 8–10, 13–17 Hydrogen bonds, 24
ethical, legal, social Hypermethylated DNA, 36
implications, 17–20 Hypomethylated DNA, 36
heavy or light, 29
hypermethylated/ Improved DNA sequencing, 19
hypomethylated, 36 Information, definition of, 1, 2
information content of, 33–38 Ionic bonds, 24
methylated, 33–34, 38
radioactive, 14–16 Life, definition of, 1
replication, 27–32 Lipids, 5
centrifugation results after, 30
models for, 28 Meselson, Matthew, 28–32
structure of, 21–27 Methyladenine, 34
DNase, 8 Methylated DNA, 33–34
44 INDEX
Methylcytosine (m5C), 34 Purines, 24

Methyltransferase, 37 Pyrimidines, 24
14
N-labeled DNA, 29 R colonies, 3–10
15
N-labeled DNA, 29 RNA, nucleotides in, 22
Nature (journal), 23 RNase, 8
Non-radioactive isotopes, 28
Nucleotide Nucleotides S colonies, 3–10
chemical composition of, 8–9 S factor, 3, 5–6
35
structure, 22 S-labeled protein, 14
Science, definition of, 1
Occam’s razor, 17 Semiconservative replication, 31
Sickle cell disease, 36
32
P-labeled DNA, 14 Silent fetal hemoglobin gene, 37
Phage protein, location after infection Stahl, Franklin, 28–32
of E. coli, 17 Streptococcus pneumonia, 3
Phage system, 14–15
Phosphates, 22 T2 phage system, 14–15
Pneumonia, 3 Thin layer chromatography
Proteases, 8 (TLC), 35
Protein, 5 Thymine, 22
aberrant, 36 Triple-stranded model, 23
disproving as heritable material,
13–20 Watson, James, 23–27
radioactive, 14–16 Wilkins, Maurice, 25–26
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The Source of Genetic

A. Malcolm Campbell, PhD

The Source of Genetic Information

All rights reserved. No part of this publication may be reproduced, stored

First published in 2016 by

ISBN-13: 978-1-94474-915-6 (print)

Momentum Press Biology Collection

Cover and interior design by S4Carlisle Publishing Services Private Ltd.,

Printed in the United States of America

Search for the Heritable

Science attempts to understand the physical world by answering ques-

heat treatment heat treatment

S cells S cells R cells S cells

S cells No cells R cells S cells

Figure 1 Griffith’s experimental design and results. S cells and R cells

protein. To test his hypothesis, Griffith tried a series of experiments that

Search for the Heritable Information 5

Based on the results from the fourth experiment in Figure 1, Griffith

Because the cell wall/membrane is composed of protein and not DNA,

1. Grind up S cells in buffer to extract all soluble material (10 mL).

Figure 2 The heritable material produces S cells from R cells.

4. Dissolve the pelleted material in 10 mL buffer.

Avery’s team precipitated the transforming S-factor after using buffer

Table 1 Comparison of four preparations of transforming factor versus

Figure 3 Chemical composition of amino acids and nucleotides. Above

anything in science. A person can accumulate large volumes of data to

Avery’s DNA solution probably did contain trace amounts of pro-

Avery OT, MacLeod CM, McCarty M. Studies on the chemical nature of

Disproving Proteins are the

normal radioactive radioactive

E. coli phage DNA

Figure 4 Hershey-Chase experimental method. A, Phage were grown

Table 2 Location of phage protein and DNA after infection of E. coli.

and two possible sources of genetic information, scientists typically choose

Ethical, Legal, Social Implications: DNA Ownership

MYTH #1: DNA sequencing is done almost instantaneously.

MYTH #2: A person’s entire DNA sequence is used in criminal cases.

MYTH #3: DNA evidence is properly used to convict criminals.

MYTH #4: DNA evidence is infallible.

DNA sequence information has many other applications beyond

20 THE SOURCE OF GENETIC INFORMATION

DNA Structure Determines

In the 1950s, biologists wanted to know the structure of DNA because,

The Structure of DNA

Figure 5 Nucleotide structures. Atoms are located at the bent corners,

category analogy chemical representation common in

Figure 6 Three types of chemical bonds. Hydrogen bonds are

DNA Structure Determines Its Function 25

experimental approach to distinguish these three models. Into this void

30 THE SOURCE OF GENETIC INFORMATION

Figure 8 Centrifugation results after DNA replication. DNA was

it will be shown that the information content in DNA is more compli-

Not all DNA Information is

It may be a surprise to learn that the information content of DNA

adenine cytosine carbon

Not all DNA Information is Linear in Nature 35

Figure 10 Two dimensional thin layer chromatography (TLC)

phosphorous to every nucleotide in the eukaryotic DNA and then sepa-

hemoglobin. The investigators wondered whether it would be possible

genotype. The extra layer of molecular epigenetic information demon-

non-radioactive isotopes. variants of atoms that do not emit radioactivity.

Methylcytosine (m5C), 34 Purines, 24