Cellular Consequences

of Evolution
Cellular Consequences
of Evolution

A. Malcolm Campbell, PhD

Christopher J. Paradise, PhD
Cellular Consequences of Evolution
Copyright A. Malcolm Campbell and Christopher J. Paradise. 2016.

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 Abstract
Once the first cell arose on Earth, how did genetic diversity arise if DNA
replication and cell division generate exact copies? The answer is that
neither process is perfect and that changes do occur at each step. Some
changes are small and subtle while others are large and dramatic. As DNA
mutates, evolution of a population takes place. But when can someone
determine if a single species has changed enough to be considered two
separate species? How is a species defined and is this definition useful in
the real world? Real biological data will be examined to confront and an-
swer these questions. Finally, the book examines an example of evolution
that takes place in humans on a regular basisthe mammalian immune
system. White blood cells evolve rapidly to confront any substance that
enters a body and is perceived as a threat. With each exposure, these cells
get better and better at neutralizing the threat.

Keywords
DNA polymerase, allele, whole genome duplication, mutation, natural
selection, single nucleotide polymorphism, dot plot, speciation, insertion,
deletion, horizontal gene transfer, GC content, provirus, copy number
variation, cancer, ploidy, paralogs, genetically modified organisms, allergy,
B cells, antibodies, secondary immune response, memory B cells, survival
signal, somatic hypermutation
 Contents
Preface...................................................................................................ix
Acknowledgments....................................................................................xi
Introduction.........................................................................................xiii
Chapter 1 The Origins of New Mutations..........................................1
Chapter 2 The Origins of New Species...............................................7
Large Scale Genome Changes............................................9
Clinical Whole Genome Changes....................................14
Genome Duplication and Speciation................................17
Ethical, Legal, Social Implications: The safety
of GMOs......................................................................19
Chapter 3 Evolution of Allergic Responses........................................23
Ethical, Legal, Social Implications: Balancing the
Rights of the Individual vs. the Group..........................29
Conclusion............................................................................................33
Glossary................................................................................................35
Index....................................................................................................37
 Preface
This book about evolution after the first cells were formed 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 about consequence of evolution at the
cellular level and some of the supporting evidence behind our under-
standing. The historic and more recent experiments and data will be
explored. Instead of believing or simply accepting information, readers of
this book will learn about the science behind evolution of cells the same
way professional scientists dowith 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.
 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. Davids 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
students learn. I benefited from the support of the Howard Hughes Med-
ical Institute grant 52006292, the James G. Martin Genomics Program,
and Davidson College. This book would not have survived its first draft
without 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
Over the last twenty years, biologists have produced reliable data and
provided a probable scenario for the origin of life on Earth. When think-
ing of evolution, most people picture dinosaurs and early human hunter
gatherers. However, evolution is not limited to animals or multicellu-
lar organisms. Evolution can happen when populations of cells change
their genotypes over time. The definition of evolution is the change in allele
frequency in a population over time. In this book, the population will
be composed of cells. The DNA content of any population varies due to
the inherent potential for DNA polymerases to make mistakes. As pre-
sented in this book, DNA is subject to many more changes than just
DNA polymerase errors. Furthermore, the human immune system has
evolved to introduce many more mutations in order to provide a better
immune response that evolves over the course of an infection.
Evolution at the cellular level can be rapid, but when a system is
well suited to meet a particular function, the system tends to evolve very
slowly if at all. As the old expression goes, If it aint broke, dont fix it.
But it is important to understand the extremes of cellular evolution, rapid
and slow, in order to discuss ethical dilemmas that face humans today.
Are genetically modified organisms (GMOs) always bad or always good?
Should peanut butter be banned from schools even when no child has suf-
fered a severe allergy attack? As a biologist, your family and community
will look to biologists for answers and it is important to understand cellu-
lar evolution in order to provide them with enough information to form
a reasoned opinion. The three chapters of this book focus on evolution at
the cellular level.
 CHAPTER 1

The Origins of New

Mutations

Meselson and Stahls classic experiment discovered that DNA uses semi-
conservative replication to make new copies. Once semiconservative
DNA replication was known, many biochemists focused their attention
on DNA polymerase to figure out how it works normally and how new
mutations occur. How can a genetic disease appear in offspring if the
parents do not have a disease allele? In other words, how do genetic mu-
tations come into being if most people dont have the disease and DNA
replication uses one strand to produce a new copy? Of course the answer
is that DNA is not always replicated faithfully, and sometimes mutations
are introduced into DNA. A mutation can be incorporated into DNA,
and once a mutation is formed, it gets replicated as faithfully as any
other portion of the DNA. Investigators wanted to better understand
how often mutations were made in DNA but they had to learn how to
polymerize DNA in vitro first.
David Baltimore and his colleague Donna Smoler wanted to know
how DNA polymerase starts the process of replication. Could DNA
polymerase start anywhere, or did it require some prior DNA on which
to add? To determine what DNA polymerase requires to start polymer-
izing, the investigators mixed equal amounts of E. coli DNA polymerase,
deoxyribonucleotide triphosphates (dNTPs), and DNA template to
three tubes. In addition to all four bases of dNTPs, the investigators
added a small amount of deoxyguanosine triphosphate (dGTP) that
contained radioactive phosphorous (32P-dGTP), which would be incor-
porated into new DNA polymers as 32P-dGMP. Each tube contained a
different amount of DNA primer as the independent variable. During
2 CELLULAR CONSEQUENCES OF EVOLUTION

the reaction, the investigators removed aliquots and quantified the

amount of radioactive dGMP incorporated (dependent variable) into
the newly-formed DNA polymers. The amount of DNA increased over
time, with more DNA formed when more primer was added. Once
they knew it was possible to initiate DNA polymerization by mixing
primers with template DNA, the investigators wanted to characterize
the chemical requirements of primers used by E. coli DNA polymerase.
They set up three reactions as before, but this time they added equal
amounts of three different types of primers. One primer was composed
of normal DNA nucleotides deoxyadenosine monophosphate (dAMP),
the second primer was composed of normal RNA nucleotides adenosine
monophosphate (AMP), and the third primer was composed of a modi-
fied dAMP with its 39 OH group removed.
The two biochemists learned that DNA polymerases require primers
to make new polymers from DNA template. The more primer available,
the more radioactive dGMP was incorporated into new DNA strands.
Under these in vitro experimental conditions, E. coli DNA polymerase
works best if the primer is a polymer of dAMP instead of AMP. Further-
more, the primer must have a 3 OH group, or the DNA polymerase
cannot add the next nucleotide onto the primer. However, subsequent
research showed that in living cells, the primer is typically composed of
RNA produced by an RNA polymerase as happens during transcription.
The distinction of RNA versus DNA primer is not as important as the
fact that all DNA polymerases require a 3' OH to add onto and they can-
not start polymerizing without a primer.
Thanks to research by Baltimore and other biochemists, investigators
got very good at conducting in vitro DNA polymerization experiments.
With their experimental capacity, biologists turned their attention to
what role DNA polymerase plays in the appearance of new mutations in
DNA. One of the first tasks was to determine if a cells DNA polymerase
had a constant rate of polymerase activity during the life of an organism.
By 1976, biologists had already discovered that non-cancerous cells have a
finite lifespan and eventually human cells die. Could the finite lifespan of
cells be caused by DNA error accumulation as a result of mutations pro-
duced by DNA polymerase? British scientists grew some human skin cells
in petri dishes and isolated DNA polymerase from an aliquot of the cells
 The Origins of New Mutations 3

right away and again later from cells that had grown for many days in the
petri dish. These two isolations yielded DNA polymerase proteins from
the human skin cells of two different ages. The investigators measured
the speed of the young versus old polymerases (Table 1). They wanted
to know if the DNA polymerases from young and old cells had the same
level of polymerase activity (quantified as units of activity).

Table 1 DNA polymerase activity comparison.

cell extracts activity
young 982 units
old 58 units
Source: From Linn et al., 1976; their table 1.

It is clear that the younger DNA polymerase was more active than
the older polymerase, but this did not necessarily mean that the younger
DNA polymerase made fewer mistakes. For example, if a student takes
an exam in 5 minutes and everyone else requires 50 minutes, the fast
students exam grade will not necessarily be 10 times higher than everyone
elses, right? An exam grade does not depend on how fast the test is com-
pleted, but how few mistakes are made. Similarly, DNA polymerases need
to be accurate more than they need to be fast. The biochemists compared
the error rate for the old and young two sources of DNA polymerase
(Table 2). They also compared young and old DNA polymerase in the pres-
ence of two different metal ions (Mg2+ and Mn2+) because it was known
that ions with a +2 charge were required for DNA polymerase activity.

Table 2 Comparison of old and young DNA polymerase

capacity.
DNA polymerase ion bases polymerized error rate
young Mg2+ 17,300 1 in 1821 bases
old Mg2+ 5,400 1 in 474 bases
young Mn2+ 26,800 1 in 1848 bases
old Mn2+ 18,800 1 in 556 bases
Source: From Linn et al., 1976; their table 2.

A few years later, a different group of biochemists wondered what

effect different +2 ions might have on human DNA polymerase ac-
curacy (Table 3). The investigators tested the effects of a few different
4 CELLULAR CONSEQUENCES OF EVOLUTION

metal ions that humans are exposed to in the environment. They com-
pared the effects of nickel (Ni2+), cadmium (Cd2+), and calcium (Ca2+)
on the accuracy of DNA polymerase isolated from young cells. They
tested at least two different concentrations of each ion to see if dosage
had an effect on the DNA polymerase.

Table 3 Comparison of ions on young human DNA

polymerase accuracy.
ions (concentration in mM) error rate
Mg2+ (1.0) 1 in 41,000
Ni2+ (1.0) 1 in 5,030
Ni2+ (2.0) 1 in 1,850
2+
Cd (0.1) 1 in 7,810
Cd2+ (0.2) 1 in 5,070
Ca2+ (0.6) 1 in 7,520
Ca2+ (1.0) 1 in 5,500
Ca2+ (2.5) 1 in 3,760

Source: From Seal et al., 1979; their table 4.

From Table 1, it is clear that older DNA polymerases generate more

mutations when DNA is replicated. In addition, it can be seen that both
young and old DNA polymerases produce more mutations in the presence
of Mg2+ than Mn2+ but without an indication of variance, it is impos-
sible to know if these values are significantly different or not. Older DNA
polymerase is affected more by the difference in ions than the young DNA
polymerase, which is consistent with younger cells containing more accu-
rate DNA polymerases. When exposed to heavy metal ions Ni2+ or Cd2+
(as found in batteries), DNA polymerases make more mutations. Table 3
illustrates why it is important to recycle old batteries rather than throw
them away. Batteries in landfills increase the odds of someone developing
cancer and the odds that children will develop new genetic diseases.
This chapter focused on how DNA replication could produce new
mutations and possibly genetic diseases. Genetic diseases are caused by
mutations in DNA sequence, which are often produced by DNA poly-
merase errors. Genomic research from 2012 has shown that each child
inherits about 100 mutations that were not present in either parent.
Therefore, mutations are common enough that everyone should think of
DNA replication as producing an equivalent DNA copy rather than an
 The Origins of New Mutations 5

exact copy. Regardless of whether the DNA polymerase produces a new

mutation or not, they all require primers with 3 OH to initiate DNA
elongation. You can search the Internet with the phrase 3D Structure
of DNA During S Phase Jsmol to see a Jsmol interactive tutorial show-
ing DNA polymerase frozen in action. When viewing the Jsmol tutorial,
try to identify which base is the template and which base is about to be
added. It is possible to see the 3 end of the last base added to the growing
DNA chain which is where the next base will be added. It is important
to see the 3 end of the growing strand, because replicating DNA grows
from this end in all species.
By studying how DNA polymerase works and contributes to muta-
tions, this chapter has presented how heritable information provides for
the continuity of life by replicating DNA. Furthermore, this chapter has
presented how inherited DNA can contain mutations which generate
variation in the population. Mutation is one of the four mechanisms of
evolution and it generates variation in a population that can be acted on
during natural selection. Double-stranded DNA uses semiconservative
replication because both strands become the template for the comple-
mentary strand. DNA polymerase requires a primer with a 3' OH group
to initiate elongation, and cells contain enzymes that produce primers as
needed. If DNA replication were perfect, it would be possible to imagine
a world with no genetic diseases and reduced variation. Chapter 2 will
present how genetics variation is beneficial and contributes to natural
selection in populations.

Bibliography
Baltimore D, Smoler D. Primer requirement and template specificity
of the DNA polymerase of RNA tumor viruses. Proc Natl Acad Sci
USA 68(7):15071511, 1971.
Kornberg T, Gefter ML. Purification and DNA synthesis in cell-free
extracts: properties of DNA polymerase II. Proc Natl Acad Sci USA
68(4):761764, 1971.
Lehman IR, Bessman MJ, Simms ES, et al. Enzymatic synthesis of deoxy-
ribonucleic acid: preparation of substrates and partial purification of
an enzyme from Escherichia coli. J Biol Chem 233(1):163170, 1958.
6 CELLULAR CONSEQUENCES OF EVOLUTION

Linn S, Kairis M, Holliday R. Decreased fidelity of DNA polymerase

activity isolated from aging human fibroblasts. Proc Natl Acad Sci
USA 73(8):28182822, 1976.
Seal G, Shearman CW, Loeb LA. On the fidelity of DNA replication:
studies with human placental DNA polymerases. J Biol Chem
254(12):52295237, 1979.
 Index
Adenosine monophosphate primer, 12
(AMP), 2 versus RNA, 2
Allergic responses replication, 1, 45
ethical, legal, social implications, template, 1
2931 Dot plot, 8
evolution of, 2331 Double-stranded DNA, 5
Antibodies, 23
antigen complex, 25 E. coli, 9, 10
first wave of, 27 pathogenic strain of, 13
primary response B cells,
produced by, 27 GC content, 1213
producing B cells, 26 Gene duplication, 11
structure and function of, 24 and speciation, 1719
Antigen-specific B cells, 2526 Genetically modified organisms
(GMOs), 1920
Baltimore, David, 12 Genetic disease, cause of, 4
Batteries, in landfills, 4 Genome duplication, 18
B cells, 23 Genomic research, 4
evolution of, 2829 Gibberellin, 7
BLAST2, 8 Golden rice, genome of, 20

Cancer cells, 15, 16 Heavy chain proteins, 23

Chisholm, Sallie W., 13 Heterozygous cells, 8
Chlamydomonas, 12 Horizontal gene transfer, 12, 13
Copy number variation, 1415 Human genome, 14
Cyanobacteria, 1314
Jsmol interactive tutorial, 5
Deoxyadenosine monophosphate
(dAMP), 2 K-12 gene, 9, 10
Deoxyguanosine triphosphate
(dGTP), 1 Le gene, 7, 8
Deoxyribonucleotide triphosphates Light chain proteins, 23
(dNTPs), 1
DNA Memory B cells, 26
deletion, 11 Mutations, origins of, 15
insertion, 11
polymerase, 15, 16 New species, origins of, 78
activity comparison, 3 clinical whole genome, changes
E. coli, 1, 2 of, 1417
ions on young human, ethical, legal, social implications
comparison of, 4 of, 1920
38 INDEX

genome duplication and speciation, Secondary immune response, 2526

1719 Single nucleotide polymorphism
large scale genome, changes (SNP), 78
of, 914 Smoler, Donna, 12
safety of GMOs, 1920 Somatic hypermutation, 27
Speciation, 9
O157:H7 gene, 9, 10 genome duplication and, 1719
Stem cells, 26
Paralogs, 17 Survival signals, 26
Pea plants, height of, 7
Ploidy number, 15 US Department of Agriculture
Primary immune response, 2526 (USDA), 31
Provirus, 13
Venter, Craig, 15
Rights of individual versus group,
balancing, 2931
RNA versus DNA primer, 2
 OTHER TITLES IN OUR BIOLOGY
COLLECTION

Cellular Structure and Functionby A. Malcolm Campbell and Christopher J. Paradise

Cellular Respirationby A. Malcolm Campbell and Christopher J. Paradise
Using DNA Information to Make Proteinsby A. Malcolm Campbell and Christopher
J.Paradise
Evolution of Eukaryotesby A. Malcolm Campbell and Christopher J. Paradise
The Source of Genetic Informationby A. Malcolm Campbell and Christopher J. Paradise
Neurons and Musclesby A. Malcolm Campbell and Christopher J. Paradise
Evolution and Origin of Cellsby A. Malcolm Campbell and Christopher J. Paradise
Reproduction and Cell Divisionby A. Malcolm Campbell and Christopher J. Paradise
Molecular Structure and Functionby A. Malcolm Campbell and Christopher J. Paradise
Animal Physiologyby A. Malcolm Campbell and Christopher J. Paradise
Cell Networksby A. Malcolm Campbell and Christopher J. Paradise
Molecular Switchesby A. Malcolm Campbell and Christopher J. Paradise
Photosynthesisby A. Malcolm Campbell and Christopher J. Paradise
Plant Physiologyby A. Malcolm Campbell and Christopher J. Paradise

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