CHAPTER 9

Molecular Biology

FIGURE 9.1 Dolly the sheep was the first cloned mammal.

CHAPTER OUTLINE
9.1 The Structure of DNA
9.2 DNA Replication
9.3 Transcription
9.4 Translation
9.5 How Genes Are Regulated

INTRODUCTION The three letters “DNA” have now become associated with crime solving,
paternity testing, human identification, and genetic testing. DNA can be retrieved from hair, blood,
or saliva. With the exception of identical twins, each person’s DNA is unique and it is possible to
detect differences between human beings on the basis of their unique DNA sequence.

DNA analysis has many practical applications beyond forensics and paternity testing. DNA testing
is used for tracing genealogy and identifying pathogens. In the medical field, DNA is used in
diagnostics, new vaccine development, and cancer therapy. It is now possible to determine
predisposition to many diseases by analyzing genes.

DNA is the genetic material passed from parent to offspring for all life on Earth. The technology of
molecular genetics developed in the last half century has enabled us to see deep into the history
of life to deduce the relationships between living things in ways never thought possible. It also
allows us to understand the workings of evolution in populations of organisms. Over a thousand
species have had their entire genome sequenced, and there have been thousands of individual
human genome sequences completed. These sequences will allow us to understand human
disease and the relationship of humans to the rest of the tree of life. Finally, molecular genetics
198 9 • Molecular Biology

techniques have revolutionized plant and animal breeding for human agricultural needs. All of
these advances in biotechnology depended on basic research leading to the discovery of the
structure of DNA in 1953, and the research since then that has uncovered the details of DNA
replication and the complex process leading to the expression of DNA in the form of proteins in the
cell.

9.1 The Structure of DNA

LEARNING OBJECTIVES
By the end of this section, you will be able to:
• Describe the structure of DNA
• Describe how eukaryotic and prokaryotic DNA is arranged in the cell

In the 1950s, Francis Crick and James Watson worked together at the University of Cambridge,
England, to determine the structure of DNA. Other scientists, such as Linus Pauling and Maurice
Wilkins, were also actively exploring this field. Pauling had discovered the secondary structure of
proteins using X-ray crystallography. X-ray crystallography is a method for investigating molecular
structure by observing the patterns formed by X-rays shot through a crystal of the substance. The
patterns give important information about the structure of the molecule of interest. In Wilkins’ lab,
researcher Rosalind Franklin was using X-ray crystallography to understand the structure of DNA.
Watson and Crick were able to piece together the puzzle of the DNA molecule using Franklin's
data (Figure 9.2). Watson and Crick also had key pieces of information available from other
researchers such as Chargaff’s rules. Chargaff had shown that of the four kinds of monomers
(nucleotides) present in a DNA molecule, two types were always present in equal amounts and the
remaining two types were also always present in equal amounts. This meant they were always
paired in some way. In 1962, James Watson, Francis Crick, and Maurice Wilkins were awarded the
Nobel Prize in Medicine for their work in determining the structure of DNA.

Access for free at openstax.org

 9.1 • The Structure of DNA 199

FIGURE 9.2 Scientist Rosalind Franklin discovered the X-ray diffraction pattern of DNA, which helped to elucidate its double helix structure.
(credit: modification of work by NIH)

Now let’s consider the structure of the two types of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). The building blocks of DNA are nucleotides, which are made up of three parts: a deoxyribose (5-carbon
sugar), a phosphate group, and a nitrogenous base (Figure 9.3). There are four types of nitrogenous bases in DNA.
Adenine (A) and guanine (G) are double-ringed purines, and cytosine (C) and thymine (T) are smaller, single-ringed
pyrimidines. The nucleotide is named according to the nitrogenous base it contains.
200 9 • Molecular Biology

FIGURE 9.3 (a) Each DNA nucleotide is made up of a sugar, a phosphate group, and a base. (b) Cytosine and thymine are pyrimidines.
Guanine and adenine are purines.

The phosphate group of one nucleotide bonds covalently with the sugar molecule of the next nucleotide, and so on,
forming a long polymer of nucleotide monomers. The sugar–phosphate groups line up in a “backbone” for each
single strand of DNA, and the nucleotide bases stick out from this backbone. The carbon atoms of the five-carbon
sugar are numbered clockwise from the oxygen as 1', 2', 3', 4', and 5' (1' is read as “one prime”). The phosphate
group is attached to the 5' carbon of one nucleotide and the 3' carbon of the next nucleotide. In its natural state,
each DNA molecule is actually composed of two single strands held together along their length with hydrogen bonds
between the bases.

Watson and Crick proposed that the DNA is made up of two strands that are twisted around each other to form a
right-handed helix, called a double helix. Base-pairing takes place between a purine and pyrimidine: namely, A
pairs with T, and G pairs with C. In other words, adenine and thymine are complementary base pairs, and cytosine
and guanine are also complementary base pairs. This is the basis for Chargaff’s rule; because of their
complementarity, there is as much adenine as thymine in a DNA molecule and as much guanine as cytosine.
Adenine and thymine are connected by two hydrogen bonds, and cytosine and guanine are connected by three
hydrogen bonds. The two strands are anti-parallel in nature; that is, one strand will have the 3' carbon of the sugar in
the “upward” position, whereas the other strand will have the 5' carbon in the upward position. The diameter of the
DNA double helix is uniform throughout because a purine (two rings) always pairs with a pyrimidine (one ring) and
their combined lengths are always equal. (Figure 9.4).

FIGURE 9.4 DNA (a) forms a double stranded helix, and (b) adenine pairs with thymine and cytosine pairs with guanine. (credit a:
modification of work by Jerome Walker, Dennis Myts)

The Structure of RNA

There is a second nucleic acid in all cells called ribonucleic acid, or RNA. Like DNA, RNA is a polymer of nucleotides.
Each of the nucleotides in RNA is made up of a nitrogenous base, a five-carbon sugar, and a phosphate group. In the
case of RNA, the five-carbon sugar is ribose, not deoxyribose. Ribose has a hydroxyl group at the 2' carbon, unlike

Access for free at openstax.org

 9.1 • The Structure of DNA 201

deoxyribose, which has only a hydrogen atom (Figure 9.5).

FIGURE 9.5 The difference between the ribose found in RNA and the deoxyribose found in DNA is that ribose has a hydroxyl group at the 2'
carbon.

RNA nucleotides contain the nitrogenous bases adenine, cytosine, and guanine. However, they do not contain
thymine, which is instead replaced by uracil, symbolized by a “U.” RNA exists as a single-stranded molecule rather
than a double-stranded helix. Molecular biologists have named several kinds of RNA on the basis of their function.
These include messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA)—molecules that are
involved in the production of proteins from the DNA code.

How DNA Is Arranged in the Cell

DNA is a working molecule; it must be replicated when a cell is ready to divide, and it must be “read” to produce the
molecules, such as proteins, to carry out the functions of the cell. For this reason, the DNA is protected and
packaged in very specific ways. In addition, DNA molecules can be very long. Stretched end-to-end, the DNA
molecules in a single human cell would come to a length of about 2 meters. Thus, the DNA for a cell must be
packaged in a very ordered way to fit and function within a structure (the cell) that is not visible to the naked eye.
The chromosomes of prokaryotes are much simpler than those of eukaryotes in many of their features (Figure 9.6).
Most prokaryotes contain a single, circular chromosome that is found in an area in the cytoplasm called the
nucleoid.

FIGURE 9.6 A eukaryote contains a well-defined nucleus, whereas in prokaryotes, the chromosome lies in the cytoplasm in an area called
the nucleoid.

The size of the genome in one of the most well-studied prokaryotes, Escherichia coli, is 4.6 million base pairs, which
would extend a distance of about 1.6 mm if stretched out. So how does this fit inside a small bacterial cell? The DNA
is twisted beyond the double helix in what is known as supercoiling. Some proteins are known to be involved in the
supercoiling; other proteins and enzymes help in maintaining the supercoiled structure.

Eukaryotes, whose chromosomes each consist of a linear DNA molecule, employ a different type of packing strategy
to fit their DNA inside the nucleus (Figure 9.7). Before the structure of DNA was even uncovered, Marie Maynard
Daly and Arthur E. Mirsky conducted extensive research in the 1940s and 1950s to understand the molecules and
structures in involved. At the most basic level, DNA is wrapped around proteins known as histones to form
structures called nucleosomes. The DNA is wrapped tightly around the histone core. This nucleosome is linked to
the next one by a short strand of DNA that is free of histones. This is also known as the “beads on a string”
202 9 • Molecular Biology

structure; the nucleosomes are the “beads” and the short lengths of DNA between them are the “string.” The
nucleosomes, with their DNA coiled around them, stack compactly onto each other to form a 30-nm–wide fiber. This
fiber is further coiled into a thicker and more compact structure. At the metaphase stage of mitosis, when the
chromosomes are lined up in the center of the cell, the chromosomes are at their most compacted. They are
approximately 700 nm in width, and are found in association with scaffold proteins.

In interphase, the phase of the cell cycle between mitoses at which the chromosomes are decondensed, eukaryotic
chromosomes have two distinct regions that can be distinguished by staining. There is a tightly packaged region that
stains darkly, and a less dense region. The darkly staining regions usually contain genes that are not active, and are
found in the regions of the centromere and telomeres. The lightly staining regions usually contain genes that are
active, with DNA packaged around nucleosomes but not further compacted.

FIGURE 9.7 These figures illustrate the compaction of the eukaryotic chromosome.

LINK TO LEARNING
Watch this animation (http://openstax.org/l/DNA_packaging) of DNA packaging.

9.2 DNA Replication

LEARNING OBJECTIVES
By the end of this section, you will be able to:
• Explain the process of DNA replication
• Explain the importance of telomerase to DNA replication
• Describe mechanisms of DNA repair

Access for free at openstax.org

 9.2 • DNA Replication 203

When a cell divides, it is important that each daughter cell receives an identical copy of the DNA. This is
accomplished by the process of DNA replication. The replication of DNA occurs during the synthesis phase, or S
phase, of the cell cycle, before the cell enters mitosis or meiosis.

The elucidation of the structure of the double helix provided a hint as to how DNA is copied. Recall that adenine
nucleotides pair with thymine nucleotides, and cytosine with guanine. This means that the two strands are
complementary to each other. For example, a strand of DNA with a nucleotide sequence of AGTCATGA will have a
complementary strand with the sequence TCAGTACT (Figure 9.8).

FIGURE 9.8 The two strands of DNA are complementary, meaning the sequence of bases in one strand can be used to create the correct
sequence of bases in the other strand.

Because of the complementarity of the two strands, having one strand means that it is possible to recreate the other
strand. This model for replication suggests that the two strands of the double helix separate during replication, and
each strand serves as a template from which the new complementary strand is copied (Figure 9.9).
204 9 • Molecular Biology

FIGURE 9.9 The semiconservative model of DNA replication is shown. Gray indicates the original DNA strands, and blue indicates newly
synthesized DNA.

During DNA replication, each of the two strands that make up the double helix serves as a template from which new
strands are copied. The new strand will be complementary to the parental or “old” strand. Each new double strand
consists of one parental strand and one new daughter strand. This is known as semiconservative replication. When
two DNA copies are formed, they have an identical sequence of nucleotide bases and are divided equally into two
daughter cells.

DNA Replication in Eukaryotes

Because eukaryotic genomes are very complex, DNA replication is a very complicated process that involves several
enzymes and other proteins. It occurs in three main stages: initiation, elongation, and termination.

Recall that eukaryotic DNA is bound to proteins known as histones to form structures called nucleosomes. During
initiation, the DNA is made accessible to the proteins and enzymes involved in the replication process. How does the
replication machinery know where on the DNA double helix to begin? It turns out that there are specific nucleotide
sequences called origins of replication at which replication begins. Certain proteins bind to the origin of replication
while an enzyme called helicase unwinds and opens up the DNA helix. As the DNA opens up, Y-shaped structures
called replication forks are formed (Figure 9.10). Two replication forks are formed at the origin of replication, and
these get extended in both directions as replication proceeds. There are multiple origins of replication on the
eukaryotic chromosome, such that replication can occur simultaneously from several places in the genome.

During elongation, an enzyme called DNA polymerase adds DNA nucleotides to the 3' end of the template. Because
DNA polymerase can only add new nucleotides at the end of a backbone, a primer sequence, which provides this
starting point, is added with complementary RNA nucleotides. This primer is removed later, and the nucleotides are
replaced with DNA nucleotides. One strand, which is complementary to the parental DNA strand, is synthesized
continuously toward the replication fork so the polymerase can add nucleotides in this direction. This continuously
synthesized strand is known as the leading strand. Because DNA polymerase can only synthesize DNA in a 5' to 3'
direction, the other new strand is put together in short pieces called Okazaki fragments. The Okazaki fragments
each require a primer made of RNA to start the synthesis. The strand with the Okazaki fragments is known as the
lagging strand. As synthesis proceeds, an enzyme removes the RNA primer, which is then replaced with DNA
nucleotides, and the gaps between fragments are sealed by an enzyme called DNA ligase.

The process of DNA replication can be summarized as follows:

Access for free at openstax.org

 9.2 • DNA Replication 205

1. DNA unwinds at the origin of replication.

2. New bases are added to the complementary parental strands. One new strand is made continuously, while the
other strand is made in pieces.
3. Primers are removed, new DNA nucleotides are put in place of the primers and the backbone is sealed by DNA
ligase.

VISUAL CONNECTION

FIGURE 9.10 A replication fork is formed by the opening of the origin of replication, and helicase separates the DNA strands. An RNA primer
is synthesized, and is elongated by the DNA polymerase. On the leading strand, DNA is synthesized continuously, whereas on the lagging
strand, DNA is synthesized in short stretches. The DNA fragments are joined by DNA ligase (not shown).

You isolate a cell strain in which the joining together of Okazaki fragments is impaired and suspect that a mutation
has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?

Telomere Replication
Because eukaryotic chromosomes are linear, DNA replication comes to the end of a line in eukaryotic chromosomes.
As you have learned, the DNA polymerase enzyme can add nucleotides in only one direction. In the leading strand,
synthesis continues until the end of the chromosome is reached; however, on the lagging strand there is no place for
a primer to be made for the DNA fragment to be copied at the end of the chromosome. This presents a problem for
the cell because the ends remain unpaired, and over time these ends get progressively shorter as cells continue to
divide. The ends of the linear chromosomes are known as telomeres, which have repetitive sequences that do not
code for a particular gene. As a consequence, it is telomeres that are shortened with each round of DNA replication
instead of genes. For example, in humans, a six base-pair sequence, TTAGGG, is repeated 100 to 1000 times. The
discovery of the enzyme telomerase (Figure 9.11) helped in the understanding of how chromosome ends are
maintained. The telomerase attaches to the end of the chromosome, and complementary bases to the RNA
template are added on the end of the DNA strand. Once the lagging strand template is sufficiently elongated, DNA
polymerase can now add nucleotides that are complementary to the ends of the chromosomes. Thus, the ends of
the chromosomes are replicated.
206 9 • Molecular Biology

FIGURE 9.11 The ends of linear chromosomes are maintained by the action of the telomerase enzyme.

Telomerase is typically found to be active in germ cells, adult stem cells, and some cancer cells. For her discovery of
telomerase and its action, Elizabeth Blackburn (Figure 9.12) received the Nobel Prize for Medicine and Physiology in
2009. Later research using HeLa cells (obtained from Henrietta Lacks) confirmed that telomerase is present in
human cells. And in 2001, researchers including Diane L. Wright found that telomerase is necessary for cells in
human embryos to rapidly proliferate.

FIGURE 9.12 Elizabeth Blackburn, 2009 Nobel Laureate, was the scientist who discovered how telomerase works. (credit: U.S. Embassy,
Stockholm, Sweden)

Telomerase is not active in adult somatic cells. Adult somatic cells that undergo cell division continue to have their
telomeres shortened. This essentially means that telomere shortening is associated with aging. In 2010, scientists
found that telomerase can reverse some age-related conditions in mice, and this may have potential in regenerative
1
medicine. Telomerase-deficient mice were used in these studies; these mice have tissue atrophy, stem-cell
depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused
extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved functioning of the testes,
spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.

DNA Replication in Prokaryotes

Recall that the prokaryotic chromosome is a circular molecule with a less extensive coiling structure than eukaryotic
chromosomes. The eukaryotic chromosome is linear and highly coiled around proteins. While there are many

1 Mariella Jaskelioff, et al., “Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice,” Nature, 469
(2011):102–7.

Access for free at openstax.org

 9.2 • DNA Replication 207

similarities in the DNA replication process, these structural differences necessitate some differences in the DNA
replication process in these two life forms.

DNA replication has been extremely well-studied in prokaryotes, primarily because of the small size of the genome
and large number of variants available. Escherichia coli has 4.6 million base pairs in a single circular chromosome,
and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding
around the chromosome in both directions. This means that approximately 1000 nucleotides are added per second.
The process is much more rapid than in eukaryotes. Table 9.1 summarizes the differences between prokaryotic and
eukaryotic replications.

Differences between Prokaryotic and Eukaryotic Replications

Property Prokaryotes Eukaryotes

Origin of replication Single Multiple

Rate of replication 1000 nucleotides/s 50 to 100 nucleotides/s

Chromosome structure circular linear

Telomerase Not present Present

TABLE 9.1

LINK TO LEARNING
Click through a tutorial (http://openstax.org/l/DNA_replicatio2) on DNA replication.

DNA Repair
DNA polymerase can make mistakes while adding nucleotides. It edits the DNA by proofreading every newly added
base. Incorrect bases are removed and replaced by the correct base, and then polymerization continues (Figure
9.13a). Most mistakes are corrected during replication, although when this does not happen, the mismatch repair
mechanism is employed. Mismatch repair enzymes recognize the wrongly incorporated base and excise it from the
DNA, replacing it with the correct base (Figure 9.13b). In yet another type of repair, nucleotide excision repair, the
DNA double strand is unwound and separated, the incorrect bases are removed along with a few bases on the 5' and
3' end, and these are replaced by copying the template with the help of DNA polymerase (Figure 9.13c). Nucleotide
excision repair is particularly important in correcting thymine dimers, which are primarily caused by ultraviolet light.
In a thymine dimer, two thymine nucleotides adjacent to each other on one strand are covalently bonded to each
other rather than their complementary bases. If the dimer is not removed and repaired it will lead to a mutation.
Individuals with flaws in their nucleotide excision repair genes show extreme sensitivity to sunlight and develop skin
cancers early in life.
208 9 • Molecular Biology

FIGURE 9.13 Proofreading by DNA polymerase (a) corrects errors during replication. In mismatch repair (b), the incorrectly added base is
detected after replication. The mismatch repair proteins detect this base and remove it from the newly synthesized strand by nuclease
action. The gap is now filled with the correctly paired base. Nucleotide excision (c) repairs thymine dimers. When exposed to UV, thymines
lying adjacent to each other can form thymine dimers. In normal cells, they are excised and replaced.

Most mistakes are corrected; if they are not, they may result in a mutation—defined as a permanent change in the
DNA sequence. Mutations in repair genes may lead to serious consequences like cancer.

Access for free at openstax.org

 9.3 • Transcription 209

9.3 Transcription
LEARNING OBJECTIVES
By the end of this section, you will be able to:
• Explain the central dogma
• Explain the main steps of transcription
• Describe how eukaryotic mRNA is processed

In both prokaryotes and eukaryotes, the second function of DNA (the first was replication) is to provide the
information needed to construct the proteins necessary so that the cell can perform all of its functions. To do this,
the DNA is “read” or transcribed into an mRNA molecule. The mRNA then provides the code to form a protein by a
process called translation. Through the processes of transcription and translation, a protein is built with a specific
sequence of amino acids that was originally encoded in the DNA. This module discusses the details of transcription.

The Central Dogma: DNA Encodes RNA; RNA Encodes Protein

The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma (Figure
9.14), which states that genes specify the sequences of mRNAs, which in turn specify the sequences of proteins.

FIGURE 9.14 The central dogma states that DNA encodes RNA, which in turn encodes protein.

The copying of DNA to mRNA is relatively straightforward, with one nucleotide being added to the mRNA strand for
every complementary nucleotide read in the DNA strand. The translation to protein is more complex because groups
of three mRNA nucleotides correspond to one amino acid of the protein sequence. However, as we shall see in the
next module, the translation to protein is still systematic, such that nucleotides 1 to 3 correspond to amino acid 1,
nucleotides 4 to 6 correspond to amino acid 2, and so on.

Transcription: from DNA to mRNA

Both prokaryotes and eukaryotes perform fundamentally the same process of transcription, with the important
difference of the membrane-bound nucleus in eukaryotes. With the genes bound in the nucleus, transcription
occurs in the nucleus of the cell and the mRNA transcript must be transported to the cytoplasm. The prokaryotes,
which include bacteria and archaea, lack membrane-bound nuclei and other organelles, and transcription occurs in
the cytoplasm of the cell. In both prokaryotes and eukaryotes, transcription occurs in three main stages: initiation,
elongation, and termination.

Initiation
Transcription requires the DNA double helix to partially unwind in the region of mRNA synthesis. The region of
unwinding is called a transcription bubble. The DNA sequence onto which the proteins and enzymes involved in
transcription bind to initiate the process is called a promoter. In most cases, promoters exist upstream of the genes
they regulate. The specific sequence of a promoter is very important because it determines whether the
corresponding gene is transcribed all of the time, some of the time, or hardly at all (Figure 9.15).
210 9 • Molecular Biology

FIGURE 9.15 The initiation of transcription begins when DNA is unwound, forming a transcription bubble. Enzymes and other proteins
involved in transcription bind at the promoter.

Elongation
Transcription always proceeds from one of the two DNA strands, which is called the template strand. The mRNA
product is complementary to the template strand and is almost identical to the other DNA strand, called the
nontemplate strand, with the exception that RNA contains a uracil (U) in place of the thymine (T) found in DNA.
During elongation, an enzyme called RNA polymerase proceeds along the DNA template adding nucleotides by
base pairing with the DNA template in a manner similar to DNA replication, with the difference that an RNA strand is
being synthesized that does not remain bound to the DNA template. As elongation proceeds, the DNA is
continuously unwound ahead of the core enzyme and rewound behind it (Figure 9.16).

FIGURE 9.16 During elongation, RNA polymerase tracks along the DNA template, synthesizes mRNA in the 5' to 3' direction, and unwinds
then rewinds the DNA as it is read.

Termination
Once a gene is transcribed, the prokaryotic polymerase needs to be instructed to dissociate from the DNA template
and liberate the newly made mRNA. Depending on the gene being transcribed, there are two kinds of termination
signals, but both involve repeated nucleotide sequences in the DNA template that result in RNA polymerase stalling,
leaving the DNA template, and freeing the mRNA transcript.

On termination, the process of transcription is complete. In a prokaryotic cell, by the time termination occurs, the
transcript would already have been used to partially synthesize numerous copies of the encoded protein because
these processes can occur concurrently using multiple ribosomes (polyribosomes) (Figure 9.17). In contrast, the
presence of a nucleus in eukaryotic cells precludes simultaneous transcription and translation.

Access for free at openstax.org

 9.3 • Transcription 211

FIGURE 9.17 Multiple polymerases can transcribe a single bacterial gene while numerous ribosomes concurrently translate the mRNA
transcripts into polypeptides. In this way, a specific protein can rapidly reach a high concentration in the bacterial cell.

Eukaryotic RNA Processing

The newly transcribed eukaryotic mRNAs must undergo several processing steps before they can be transferred
from the nucleus to the cytoplasm and translated into a protein. The additional steps involved in eukaryotic mRNA
maturation create a molecule that is much more stable than a prokaryotic mRNA. For example, eukaryotic mRNAs
last for several hours, whereas the typical prokaryotic mRNA lasts no more than five seconds.

The mRNA transcript is first coated in RNA-stabilizing proteins to prevent it from degrading while it is processed and
exported out of the nucleus. This occurs while the pre-mRNA still is being synthesized by adding a special
nucleotide “cap” to the 5' end of the growing transcript. In addition to preventing degradation, factors involved in
protein synthesis recognize the cap to help initiate translation by ribosomes.

Once elongation is complete, an enzyme then adds a string of approximately 200 adenine residues to the 3' end,
called the poly-A tail. This modification further protects the pre-mRNA from degradation and signals to cellular
factors that the transcript needs to be exported to the cytoplasm.

Eukaryotic genes are composed of protein-coding sequences called exons (ex-on signifies that they are expressed)
and intervening sequences called introns (int-ron denotes their intervening role). Introns are removed from the pre-
mRNA during processing. Intron sequences in mRNA do not encode functional proteins. It is essential that all of a
pre-mRNA’s introns be completely and precisely removed before protein synthesis so that the exons join together to
code for the correct amino acids. If the process errs by even a single nucleotide, the sequence of the rejoined exons
would be shifted, and the resulting protein would be nonfunctional. The process of removing introns and
reconnecting exons is called splicing (Figure 9.18). Introns are removed and degraded while the pre-mRNA is still in
the nucleus.

FIGURE 9.18 Eukaryotic mRNA contains introns that must be spliced out. A 5' cap and 3' tail are also added.
212 9 • Molecular Biology

9.4 Translation
LEARNING OBJECTIVES
By the end of this section, you will be able to:
• Describe the different steps in protein synthesis
• Discuss the role of ribosomes in protein synthesis
• Describe the genetic code and how the nucleotide sequence determines the amino acid and the protein
sequence

The synthesis of proteins is one of a cell’s most energy-consuming metabolic processes. In turn, proteins account
for more mass than any other component of living organisms (with the exception of water), and proteins perform a
wide variety of the functions of a cell. The process of translation, or protein synthesis, involves decoding an mRNA
message into a polypeptide product. Amino acids are covalently strung together in lengths ranging from
approximately 50 amino acids to more than 1,000.

The Protein Synthesis Machinery

In addition to the mRNA template, many other molecules contribute to the process of translation. The composition
of each component may vary across species; for instance, ribosomes may consist of different numbers of ribosomal
RNAs (rRNA) and polypeptides depending on the organism. However, the general structures and functions of the
protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an
mRNA template, ribosomes, tRNAs, and various enzymatic factors (Figure 9.19).

FIGURE 9.19 The protein synthesis machinery includes the large and small subunits of the ribosome, mRNA, and tRNA. (credit:
modification of work by NIGMS, NIH)

In E. coli, there are 200,000 ribosomes present in every cell at any given time. A ribosome is a complex
macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the
nucleolus is completely specialized for the synthesis and assembly of rRNAs.

Ribosomes are located in the cytoplasm in prokaryotes and in the cytoplasm and endoplasmic reticulum of
eukaryotes. Ribosomes are made up of a large and a small subunit that come together for translation. The small
subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs, a type
of RNA molecule that brings amino acids to the growing chain of the polypeptide. Each mRNA molecule is
simultaneously translated by many ribosomes, all synthesizing protein in the same direction.

Depending on the species, 40 to 60 types of tRNA exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to
sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs
are the molecules that actually “translate” the language of RNA into the language of proteins. For each tRNA to
function, it must have its specific amino acid bonded to it. In the process of tRNA “charging,” each tRNA molecule is
bonded to its correct amino acid.

Access for free at openstax.org

 9.4 • Translation 213

The Genetic Code

To summarize what we know to this point, the cellular process of transcription generates messenger RNA (mRNA), a
mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the mRNA
template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20
commonly occurring amino acids; therefore, it can be said that the protein alphabet consists of 20 letters. Each
amino acid is defined by a three-nucleotide sequence called the triplet codon. The relationship between a
nucleotide codon and its corresponding amino acid is called the genetic code.

Given the different numbers of “letters” in the mRNA and protein “alphabets,” combinations of nucleotides
corresponded to single amino acids. Using a three-nucleotide code means that there are a total of 64 (4 × 4 × 4)
possible combinations; therefore, a given amino acid is encoded by more than one nucleotide triplet (Figure 9.20).

FIGURE 9.20 This figure shows the genetic code for translating each nucleotide triplet, or codon, in mRNA into an amino acid or a
termination signal in a nascent protein. (credit: modification of work by NIH)

Three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery.
These triplets are called stop codons. Another codon, AUG, also has a special function. In addition to specifying the
amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is
set by the AUG start codon near the 5' end of the mRNA. The genetic code is universal. With a few exceptions,
virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all life on Earth
shares a common origin.

The Mechanism of Protein Synthesis

Just as with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and
termination. The process of translation is similar in prokaryotes and eukaryotes. Here we will explore how
translation occurs in E. coli, a representative prokaryote, and specify any differences between prokaryotic and
eukaryotic translation.

Protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small
ribosome subunit, the mRNA template, three initiation factors, and a special initiator tRNA. The initiator tRNA
interacts with the AUG start codon, and links to a special form of the amino acid methionine that is typically
removed from the polypeptide after translation is complete.

In prokaryotes and eukaryotes, the basics of polypeptide elongation are the same, so we will review elongation from
the perspective of E. coli. The large ribosomal subunit of E. coli consists of three compartments: the A site binds
incoming charged tRNAs (tRNAs with their attached specific amino acids). The P site binds charged tRNAs carrying
amino acids that have formed bonds with the growing polypeptide chain but have not yet dissociated from their
corresponding tRNA. The E site releases dissociated tRNAs so they can be recharged with free amino acids. The
214 9 • Molecular Biology

ribosome shifts one codon at a time, catalyzing each process that occurs in the three sites. With each step, a
charged tRNA enters the complex, the polypeptide becomes one amino acid longer, and an uncharged tRNA departs.
The energy for each bond between amino acids is derived from GTP, a molecule similar to ATP (Figure 9.21).
Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a
200-amino acid polypeptide could be translated in just 10 seconds.

FIGURE 9.21 Translation begins when a tRNA anticodon recognizes a codon on the mRNA. The large ribosomal subunit joins the small
subunit, and a second tRNA is recruited. As the mRNA moves relative to the ribosome, the polypeptide chain is formed. Entry of a release
factor into the A site terminates translation and the components dissociate.

Termination of translation occurs when a stop codon (UAA, UAG, or UGA) is encountered. When the ribosome
encounters the stop codon, the growing polypeptide is released and the ribosome subunits dissociate and leave the
mRNA. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused
in another transcription reaction.

LINK TO LEARNING
Transcribe a gene and translate it to protein using complementary pairing and the genetic code at this site
(http://openstax.org/l/create_protein2).

Access for free at openstax.org

 9.5 • How Genes Are Regulated 215

9.5 How Genes Are Regulated

LEARNING OBJECTIVES
By the end of this section, you will be able to:
• Discuss why every cell does not express all of its genes
• Describe how prokaryotic gene expression occurs at the transcriptional level
• Understand that eukaryotic gene expression occurs at the epigenetic, transcriptional, post-
transcriptional, translational, and post-translational levels

For a cell to function properly, necessary proteins must be synthesized at the proper time. All organisms and cells
control or regulate the transcription and translation of their DNA into protein. The process of turning on a gene to
produce RNA and protein is called gene expression. Whether in a simple unicellular organism or in a complex
multicellular organism, each cell controls when and how its genes are expressed. For this to occur, there must be a
mechanism to control when a gene is expressed to make RNA and protein, how much of the protein is made, and
when it is time to stop making that protein because it is no longer needed.

Cells in multicellular organisms are specialized; cells in different tissues look very different and perform different
functions. For example, a muscle cell is very different from a liver cell, which is very different from a skin cell. These
differences are a consequence of the expression of different sets of genes in each of these cells. All cells have
certain basic functions they must perform for themselves, such as converting the energy in sugar molecules into
energy in ATP. Each cell also has many genes that are not expressed, and expresses many that are not expressed by
other cells, such that it can carry out its specialized functions. In addition, cells will turn on or off certain genes at
different times in response to changes in the environment or at different times during the development of the
organism. Unicellular organisms, both eukaryotic and prokaryotic, also turn on and off genes in response to the
demands of their environment so that they can respond to special conditions.

The control of gene expression is extremely complex. Malfunctions in this process are detrimental to the cell and
can lead to the development of many diseases, including cancer.

Prokaryotic versus Eukaryotic Gene Expression

To understand how gene expression is regulated, we must first understand how a gene becomes a functional protein
in a cell. The process occurs in both prokaryotic and eukaryotic cells, just in slightly different fashions.

Because prokaryotic organisms lack a cell nucleus, the processes of transcription and translation occur almost
simultaneously. When the protein is no longer needed, transcription stops. As a result, the primary method to
control what type and how much protein is expressed in a prokaryotic cell is through the regulation of DNA
transcription into RNA. All the subsequent steps happen automatically. When more protein is required, more
transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is almost entirely at the
transcriptional level.

The first example of such control was discovered using E. coli in the 1950s and 1960s by French researchers and is
called the lac operon. The lac operon is a stretch of DNA with three adjacent genes that code for proteins that
participate in the absorption and metabolism of lactose, a food source for E. coli. When lactose is not present in the
bacterium’s environment, the lac genes are transcribed in small amounts. When lactose is present, the genes are
transcribed and the bacterium is able to use the lactose as a food source. The operon also contains a promoter
sequence to which the RNA polymerase binds to begin transcription; between the promoter and the three genes is a
region called the operator. When there is no lactose present, a protein known as a repressor binds to the operator
and prevents RNA polymerase from binding to the promoter, except in rare cases. Thus very little of the protein
products of the three genes is made. When lactose is present, an end product of lactose metabolism binds to the
repressor protein and prevents it from binding to the operator. This allows RNA polymerase to bind to the promoter
and freely transcribe the three genes, allowing the organism to metabolize the lactose.

Eukaryotic cells, in contrast, have intracellular organelles and are much more complex. Recall that in eukaryotic
cells, the DNA is contained inside the cell’s nucleus and it is transcribed into mRNA there. The newly synthesized
mRNA is then transported out of the nucleus into the cytoplasm, where ribosomes translate the mRNA into protein.
The processes of transcription and translation are physically separated by the nuclear membrane; transcription
occurs only within the nucleus, and translation only occurs outside the nucleus in the cytoplasm. The regulation of
216 9 • Molecular Biology

gene expression can occur at all stages of the process (Figure 9.22). Regulation may occur when the DNA is uncoiled
and loosened from nucleosomes to bind transcription factors (epigenetic level), when the RNA is transcribed
(transcriptional level), when RNA is processed and exported to the cytoplasm after it is transcribed (post-
transcriptional level), when the RNA is translated into protein (translational level), or after the protein has been
made (post-translational level).

FIGURE 9.22 Eukaryotic gene expression is regulated during transcription and RNA processing, which take place in the nucleus, as well as
during protein translation, which takes place in the cytoplasm. Further regulation may occur through post-translational modifications of
proteins.

The differences in the regulation of gene expression between prokaryotes and eukaryotes are summarized in Table
9.2.

Access for free at openstax.org

 9.5 • How Genes Are Regulated 217

Differences in the Regulation of Gene Expression of Prokaryotic and Eukaryotic Organisms

Prokaryotic organisms Eukaryotic organisms

Lack nucleus Contain nucleus

• RNA transcription occurs prior to protein translation, and it takes place

RNA transcription and protein in the nucleus. RNA translation to protein occurs in the cytoplasm.
translation occur almost • RNA post-processing includes addition of a 5' cap, poly-A tail, and
simultaneously excision of introns and splicing of exons.

Gene expression is regulated Gene expression is regulated at many levels (epigenetic, transcriptional,
primarily at the transcriptional level post-transcriptional, translational, and post-translational)

TABLE 9.2

EVOLUTION CONNECTION

Alternative RNA Splicing

In the 1970s, genes were first observed that exhibited alternative RNA splicing. Alternative RNA splicing is a
mechanism that allows different protein products to be produced from one gene when different combinations of
introns (and sometimes exons) are removed from the transcript (Figure 9.23). This alternative splicing can be
haphazard, but more often it is controlled and acts as a mechanism of gene regulation, with the frequency of
different splicing alternatives controlled by the cell as a way to control the production of different protein products
in different cells, or at different stages of development. Alternative splicing is now understood to be a common
mechanism of gene regulation in eukaryotes; according to one estimate, 70% of genes in humans are expressed as
multiple proteins through alternative splicing.
218 9 • Molecular Biology

FIGURE 9.23 There are five basic modes of alternative splicing. Segments of pre-mRNA with exons shown in blue, red, orange, and pink can
be spliced to produce a variety of new mature mRNA segments.

How could alternative splicing evolve? Introns have a beginning and ending recognition sequence, and it is easy to
imagine the failure of the splicing mechanism to identify the end of an intron and find the end of the next intron, thus
removing two introns and the intervening exon. In fact, there are mechanisms in place to prevent such exon
skipping, but mutations are likely to lead to their failure. Such “mistakes” would more than likely produce a
nonfunctional protein. Indeed, the cause of many genetic diseases is alternative splicing rather than mutations in a
sequence. However, alternative splicing would create a protein variant without the loss of the original protein,
opening up possibilities for adaptation of the new variant to new functions. Gene duplication has played an
important role in the evolution of new functions in a similar way—by providing genes that may evolve without
eliminating the original functional protein.

Access for free at openstax.org

 9 • Key Terms 219

Key Terms
alternative RNA splicing a post-transcriptional gene nontemplate strand the strand of DNA that is not
regulation mechanism in eukaryotes in which used to transcribe mRNA; this strand is identical to
multiple protein products are produced by a single the mRNA except that T nucleotides in the DNA are
gene through alternative splicing combinations of replaced by U nucleotides in the mRNA
the RNA transcript nucleotide excision repair a form of DNA repair in
codon three consecutive nucleotides in mRNA that which the DNA molecule is unwound and separated
specify the addition of a specific amino acid or the in the region of the nucleotide damage, the
release of a polypeptide chain during translation damaged nucleotides are removed and replaced
deoxyribose a five-carbon sugar molecule with a with new nucleotides using the complementary
hydrogen atom rather than a hydroxyl group in the strand, and the DNA strand is resealed and allowed
2' position; the sugar component of DNA to rejoin its complement
nucleotides Okazaki fragments the DNA fragments that are
DNA ligase the enzyme that catalyzes the joining of synthesized in short stretches on the lagging strand
DNA fragments together phosphate group a molecular group consisting of a
DNA polymerase an enzyme that synthesizes a new central phosphorus atom bound to four oxygen
strand of DNA complementary to a template strand atoms
double helix the molecular shape of DNA in which post-transcriptional control of gene expression after
two strands of nucleotides wind around each other the RNA molecule has been created but before it is
in a spiral shape translated into protein
epigenetic describing non-genetic regulatory factors, post-translational control of gene expression after a
such as changes in modifications to histone proteins protein has been created
and DNA that control accessibility to genes in primer a short stretch of RNA nucleotides that is
chromosomes required to initiate replication and allow DNA
exon a sequence present in protein-coding mRNA polymerase to bind and begin replication
after completion of pre-mRNA splicing promoter a sequence on DNA to which RNA
gene expression processes that control whether a polymerase and associated factors bind and initiate
gene is expressed transcription
genetic code the amino acids that correspond to replication fork the Y-shaped structure formed
three-nucleotide codons of mRNA during the initiation of replication
helicase an enzyme that helps to open up the DNA RNA polymerase an enzyme that synthesizes an RNA
helix during DNA replication by breaking the strand from a DNA template strand
hydrogen bonds rRNA ribosomal RNA; molecules of RNA that combine
intron non–protein-coding intervening sequences to form part of the ribosome
that are spliced from mRNA during processing semiconservative replication the method used to
lagging strand during replication of the 3' to 5' replicate DNA in which the double-stranded
strand, the strand that is replicated in short molecule is separated and each strand acts as a
fragments and away from the replication fork template for a new strand to be synthesized, so the
leading strand the strand that is synthesized resulting DNA molecules are composed of one new
continuously in the 5' to 3' direction that is strand of nucleotides and one old strand of
synthesized in the direction of the replication fork nucleotides
mismatch repair a form of DNA repair in which non- splicing the process of removing introns and
complementary nucleotides are recognized, reconnecting exons in a pre-mRNA
excised, and replaced with correct nucleotides start codon the AUG (or, rarely GUG) on an mRNA
mRNA messenger RNA; a form of RNA that carries from which translation begins; always specifies
the nucleotide sequence code for a protein methionine
sequence that is translated into a polypeptide stop codon one of the three mRNA codons that
sequence specifies termination of translation
mutation a permanent variation in the nucleotide telomerase an enzyme that contains a catalytic part
sequence of a genome and an inbuilt RNA template; it functions to maintain
nitrogenous base a nitrogen-containing molecule telomeres at chromosome ends
that acts as a base; often referring to one of the telomere the DNA at the end of linear chromosomes
purine or pyrimidine components of nucleic acids template strand the strand of DNA that specifies the
220 9 • Chapter Summary

complementary mRNA molecule specific three-nucleotide anticodon sequence to

transcription bubble the region of locally unwound pair with the mRNA codon and also binds to a
DNA that allows for transcription of mRNA specific amino acid
tRNA transfer RNA; an RNA molecule that contains a

Chapter Summary
9.1 The Structure of DNA a single origin of replication. The DNA is opened with
enzymes, resulting in the formation of the replication
The model of the double-helix structure of DNA was
fork. Primase synthesizes an RNA primer to initiate
proposed by Watson and Crick. The DNA molecule is a
synthesis by DNA polymerase, which can add
polymer of nucleotides. Each nucleotide is composed
nucleotides in only one direction. One strand is
of a nitrogenous base, a five-carbon sugar
synthesized continuously in the direction of the
(deoxyribose), and a phosphate group. There are four
replication fork; this is called the leading strand. The
nitrogenous bases in DNA, two purines (adenine and
other strand is synthesized in a direction away from the
guanine) and two pyrimidines (cytosine and thymine).
replication fork, in short stretches of DNA known as
A DNA molecule is composed of two strands. Each
Okazaki fragments. This strand is known as the lagging
strand is composed of nucleotides bonded together
strand. Once replication is completed, the RNA primers
covalently between the phosphate group of one and
are replaced by DNA nucleotides and the DNA is sealed
the deoxyribose sugar of the next. From this backbone
with DNA ligase.
extend the bases. The bases of one strand bond to the
bases of the second strand with hydrogen bonds. The ends of eukaryotic chromosomes pose a problem,
Adenine always bonds with thymine, and cytosine as polymerase is unable to extend them without a
always bonds with guanine. The bonding causes the primer. Telomerase, an enzyme with an inbuilt RNA
two strands to spiral around each other in a shape template, extends the ends by copying the RNA
called a double helix. Ribonucleic acid (RNA) is a template and extending one end of the chromosome.
second nucleic acid found in cells. RNA is a single- DNA polymerase can then extend the DNA using the
stranded polymer of nucleotides. It also differs from primer. In this way, the ends of the chromosomes are
DNA in that it contains the sugar ribose, rather than protected. Cells have mechanisms for repairing DNA
deoxyribose, and the nucleotide uracil rather than when it becomes damaged or errors are made in
thymine. Various RNA molecules function in the replication. These mechanisms include mismatch
process of forming proteins from the genetic code in repair to replace nucleotides that are paired with a
DNA. non-complementary base and nucleotide excision
repair, which removes bases that are damaged such as
Prokaryotes contain a single, double-stranded circular
thymine dimers.
chromosome. Eukaryotes contain double-stranded
linear DNA molecules packaged into chromosomes. 9.3 Transcription
The DNA helix is wrapped around proteins to form
In prokaryotes, mRNA synthesis is initiated at a
nucleosomes. The protein coils are further coiled, and
promoter sequence on the DNA template. Elongation
during mitosis and meiosis, the chromosomes become
synthesizes new mRNA. Termination liberates the
even more greatly coiled to facilitate their movement.
mRNA and occurs by mechanisms that stall the RNA
Chromosomes have two distinct regions which can be
polymerase and cause it to fall off the DNA template.
distinguished by staining, reflecting different degrees
Newly transcribed eukaryotic mRNAs are modified with
of packaging and determined by whether the DNA in a
a cap and a poly-A tail. These structures protect the
region is being expressed (euchromatin) or not
mature mRNA from degradation and help export it from
(heterochromatin).
the nucleus. Eukaryotic mRNAs also undergo splicing,
9.2 DNA Replication in which introns are removed and exons are
reconnected with single-nucleotide accuracy. Only
DNA replicates by a semi-conservative method in
finished mRNAs are exported from the nucleus to the
which each of the two parental DNA strands act as a
cytoplasm.
template for new DNA to be synthesized. After
replication, each DNA has one parental or “old” strand, 9.4 Translation
and one daughter or “new” strand.
The central dogma describes the flow of genetic
Replication in eukaryotes starts at multiple origins of information in the cell from genes to mRNA to proteins.
replication, while replication in prokaryotes starts from Genes are used to make mRNA by the process of

Access for free at openstax.org

 9 • Visual Connection Questions 221

transcription; mRNA is used to synthesize proteins by dissociates the components and frees the new protein.
the process of translation. The genetic code is the
correspondence between the three-nucleotide mRNA
9.5 How Genes Are Regulated
codon and an amino acid. The genetic code is While all somatic cells within an organism contain the
“translated” by the tRNA molecules, which associate a same DNA, not all cells within that organism express
specific codon with a specific amino acid. The genetic the same proteins. Prokaryotic organisms express the
code is degenerate because 64 triplet codons in mRNA entire DNA they encode in every cell, but not
specify only 20 amino acids and three stop codons. necessarily all at the same time. Proteins are
This means that more than one codon corresponds to expressed only when they are needed. Eukaryotic
an amino acid. Almost every species on the planet uses organisms express a subset of the DNA that is encoded
the same genetic code. in any given cell. In each cell type, the type and amount
of protein is regulated by controlling gene expression.
The players in translation include the mRNA template,
To express a protein, the DNA is first transcribed into
ribosomes, tRNAs, and various enzymatic factors. The
RNA, which is then translated into proteins. In
small ribosomal subunit binds to the mRNA template.
prokaryotic cells, these processes occur almost
Translation begins at the initiating AUG on the mRNA.
simultaneously. In eukaryotic cells, transcription
The formation of bonds occurs between sequential
occurs in the nucleus and is separate from the
amino acids specified by the mRNA template according
translation that occurs in the cytoplasm. Gene
to the genetic code. The ribosome accepts charged
expression in prokaryotes is regulated only at the
tRNAs, and as it steps along the mRNA, it catalyzes
transcriptional level, whereas in eukaryotic cells, gene
bonding between the new amino acid and the end of
expression is regulated at the epigenetic,
the growing polypeptide. The entire mRNA is translated
transcriptional, post-transcriptional, translational, and
in three-nucleotide “steps” of the ribosome. When a
post-translational levels.
stop codon is encountered, a release factor binds and

Visual Connection Questions

1. Figure 9.10 You isolate a cell strain in which the
joining together of Okazaki fragments is impaired
and suspect that a mutation has occurred in an
enzyme found at the replication fork. Which enzyme
is most likely to be mutated?

Review Questions
2. Which of the following does cytosine pair with? 5. The initial mechanism for repairing nucleotide
a. guanine errors in DNA is ________.
b. thymine a. mismatch repair
c. adenine b. DNA polymerase proofreading
d. a pyrimidine c. nucleotide excision repair
d. thymine dimers
3. Prokaryotes contain a ________chromosome, and
eukaryotes contain ________ chromosomes. 6. A promoter is ________.
a. single-stranded circular; single-stranded linear a. a specific sequence of DNA nucleotides
b. single-stranded linear; single-stranded circular b. a specific sequence of RNA nucleotides
c. double-stranded circular; double-stranded c. a protein that binds to DNA
linear d. an enzyme that synthesizes RNA
d. double-stranded linear; double-stranded
circular 7. Portions of eukaryotic mRNA sequence that are
removed during RNA processing are ________.
4. DNA replicates by which of the following models? a. exons
a. conservative b. caps
b. semiconservative c. poly-A tails
c. dispersive d. introns
d. none of the above
222 9 • Critical Thinking Questions

8. The RNA components of ribosomes are synthesized 10. Control of gene expression in eukaryotic cells
in the ________. occurs at which level(s)?
a. cytoplasm a. only the transcriptional level
b. nucleus b. epigenetic and transcriptional levels
c. nucleolus c. epigenetic, transcriptional, and translational
d. endoplasmic reticulum levels
d. epigenetic, transcriptional, post-
9. How long would the peptide be that is translated transcriptional, translational, and post-
from this MRNA sequence: 5'-AUGGGCUACCGA-3'? translational levels
a. 0
b. 2 11. Post-translational control refers to:
c. 3 a. regulation of gene expression after
d. 4 transcription
b. regulation of gene expression after translation
c. control of epigenetic activation
d. period between transcription and translation

Critical Thinking Questions

12. Describe the organization of the eukaryotic 15. Transcribe and translate the following DNA
chromosome. sequence (nontemplate strand): 5'-
ATGGCCGGTTATTAAGCA-3'
13. Describe the structure and complementary base
pairing of DNA. 16. Describe how controlling gene expression will
alter the overall protein levels in the cell.
14. How do the linear chromosomes in eukaryotes
ensure that its ends are replicated completely?

Access for free at openstax.org

CHAPTER 10
Biotechnology

FIGURE 10.1 (a) A thermal cycler, such as the one shown here, is a basic tool used to study DNA in a process called
the polymerase chain reaction (PCR). The polymerase enzyme most often used with PCR comes from a strain of
bacteria that lives in (b) the hot springs of Yellowstone National Park. (credit a: modification of work by Magnus
Manske; credit b: modification of work by Jon Sullivan)

CHAPTER OUTLINE
10.1 Cloning and Genetic Engineering
10.2 Biotechnology in Medicine and Agriculture
10.3 Genomics and Proteomics

INTRODUCTION The latter half of the twentieth century began with the discovery of the structure
of DNA, then progressed to the development of the basic tools used to study and manipulate DNA.
These advances, as well as advances in our understanding of and ability to manipulate cells, have
led some to refer to the twenty-first century as the biotechnology century. The rate of discovery
and of the development of new applications in medicine, agriculture, and energy is expected to
accelerate, bringing huge benefits to humankind and perhaps also significant risks. Many of these
developments are expected to raise significant ethical and social questions that human societies
have not yet had to consider.

10.1 Cloning and Genetic Engineering

LEARNING OBJECTIVES
By the end of this section, you will be able to:
• Explain the basic techniques used to manipulate genetic material
• Explain molecular and reproductive cloning

Biotechnology is the use of artificial methods to modify the genetic material of living organisms or
cells to produce novel compounds or to perform new functions. Biotechnology has been used for
improving livestock and crops since the beginning of agriculture through selective breeding. Since
the discovery of the structure of DNA in 1953, and particularly since the development of tools and
methods to manipulate DNA in the 1970s, biotechnology has become synonymous with the
manipulation of organisms’ DNA at the molecular level. The primary applications of this
technology are in medicine (for the production of vaccines and antibiotics) and in agriculture (for
224 10 • Biotechnology

the genetic modification of crops). Biotechnology also has many industrial applications, such as
fermentation, the treatment of oil spills, and the production of biofuels, as well as many
household applications such as the use of enzymes in laundry detergent.

Manipulating Genetic Material

To accomplish the applications described above, biotechnologists must be able to extract,
manipulate, and analyze nucleic acids.

Review of Nucleic Acid Structure

To understand the basic techniques used to work with nucleic acids, remember that nucleic acids
are macromolecules made of nucleotides (a sugar, a phosphate, and a nitrogenous base). The
phosphate groups on these molecules each have a net negative charge. An entire set of DNA
molecules in the nucleus of eukaryotic organisms is called the genome. DNA has two
complementary strands linked by hydrogen bonds between the paired bases.

Unlike DNA in eukaryotic cells, RNA molecules leave the nucleus. Messenger RNA (mRNA) is
analyzed most frequently because it represents the protein-coding genes that are being expressed
in the cell.

Isolation of Nucleic Acids

To study or manipulate nucleic acids, the DNA must first be extracted from cells. Various
techniques are used to extract different types of DNA (Figure 10.2). Most nucleic acid extraction
techniques involve steps to break open the cell, and then the use of enzymatic reactions to
destroy all undesired macromolecules. Cells are broken open using a detergent solution
containing buffering compounds. To prevent degradation and contamination, macromolecules
such as proteins and RNA are inactivated using enzymes. The DNA is then brought out of solution
using alcohol. The resulting DNA, because it is made up of long polymers, forms a gelatinous
mass.

FIGURE 10.2 This diagram shows the basic method used for the extraction of DNA.

RNA is studied to understand gene expression patterns in cells. RNA is naturally very unstable
because enzymes that break down RNA are commonly present in nature. Some are even secreted
by our own skin and are very difficult to inactivate. Similar to DNA extraction, RNA extraction
involves the use of various buffers and enzymes to inactivate other macromolecules and preserve
only the RNA.

Access for free at openstax.org

 10.1 • Cloning and Genetic Engineering 225

Gel Electrophoresis
Because nucleic acids are negatively charged ions at neutral or alkaline pH in an aqueous environment, they can be
moved by an electric field. Gel electrophoresis is a technique used to separate charged molecules on the basis of
size and charge. The nucleic acids can be separated as whole chromosomes or as fragments. The nucleic acids are
loaded into a slot at one end of a gel matrix, an electric current is applied, and negatively charged molecules are
pulled toward the opposite end of the gel (the end with the positive electrode). Smaller molecules move through the
pores in the gel faster than larger molecules; this difference in the rate of migration separates the fragments on the
basis of size. The nucleic acids in a gel matrix are invisible until they are stained with a compound that allows them
to be seen, such as a dye. Distinct fragments of nucleic acids appear as bands at specific distances from the top of
the gel (the negative electrode end) that are based on their size (Figure 10.3). A mixture of many fragments of
varying sizes appear as a long smear, whereas uncut genomic DNA is usually too large to run through the gel and
forms a single large band at the top of the gel.

FIGURE 10.3 Shown are DNA fragments from six samples run on a gel, stained with a fluorescent dye and viewed under UV light. (credit:
modification of work by James Jacob, Tompkins Cortland Community College)

Polymerase Chain Reaction

DNA analysis often requires focusing on one or more specific regions of the genome. It also frequently involves
situations in which only one or a few copies of a DNA molecule are available for further analysis. These amounts are
insufficient for most procedures, such as gel electrophoresis. Polymerase chain reaction (PCR) is a technique used
to rapidly increase the number of copies of specific regions of DNA for further analyses (Figure 10.4). PCR uses a
special form of DNA polymerase, the enzyme that replicates DNA, and other short nucleotide sequences called
primers that base pair to a specific portion of the DNA being replicated. PCR is used for many purposes in
laboratories. These include: 1) the identification of the owner of a DNA sample left at a crime scene; 2) paternity
analysis; 3) the comparison of small amounts of ancient DNA with modern organisms; and 4) determining the
sequence of nucleotides in a specific region.
226 10 • Biotechnology

FIGURE 10.4 Polymerase chain reaction, or PCR, is used to produce many copies of a specific sequence of DNA using a special form of DNA
polymerase.

Cloning
In general, cloning means the creation of a perfect replica. Typically, the word is used to describe the creation of a
genetically identical copy. In biology, the re-creation of a whole organism is referred to as “reproductive cloning.”
Long before attempts were made to clone an entire organism, researchers learned how to copy short stretches of
DNA—a process that is referred to as molecular cloning. The technique offered methods to create new medicines
and to overcome difficulties with existing ones. When Lydia Villa-Komaroff, working in the Gilbert Lab at Harvard,
published the first paper outlining the technique for producing synthetic insulin, diabetes researchers and patients
received new hope in fighting the disease. Insulin at that time was only produced using pig and cow pancreases, and
the life-saving substance was often in short supply. Synthetic insulin, once mass produced, would solve that
problem for many patients. These early discoveries led to the "BioTech Boom," and spurred continued research and
funding for newer and better ways to improve health.

Molecular Cloning
Cloning allows for the creation of multiple copies of genes, expression of genes, and study of specific genes. To get
the DNA fragment into a bacterial cell in a form that will be copied or expressed, the fragment is first inserted into a
plasmid. A plasmid (also called a vector in this context) is a small circular DNA molecule that replicates
independently of the chromosomal DNA in bacteria. In cloning, the plasmid molecules can be used to provide a

Access for free at openstax.org

 10.1 • Cloning and Genetic Engineering 227

"vehicle" in which to insert a desired DNA fragment. Modified plasmids are usually reintroduced into a bacterial host
for replication. As the bacteria divide, they copy their own DNA (including the plasmids). The inserted DNA fragment
is copied along with the rest of the bacterial DNA. In a bacterial cell, the fragment of DNA from the human genome
(or another organism that is being studied) is referred to as foreign DNA to differentiate it from the DNA of the
bacterium (the host DNA).

Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute
favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics). Plasmids
have been highly engineered as vectors for molecular cloning and for the subsequent large-scale production of
important molecules, such as insulin. A valuable characteristic of plasmid vectors is the ease with which a foreign
DNA fragment can be introduced. These plasmid vectors contain many short DNA sequences that can be cut with
different commonly available restriction enzymes. Restriction enzymes (also called restriction endonucleases)
recognize specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as
a defense mechanism against foreign DNA. Many restriction enzymes make staggered cuts in the two strands of
DNA, such that the cut ends have a 2- to 4-nucleotide single-stranded overhang. The sequence that is recognized by
the restriction enzyme is a four- to eight-nucleotide sequence that is a palindrome. Like with a word palindrome, this
means the sequence reads the same forward and backward. In most cases, the sequence reads the same forward
on one strand and backward on the complementary strand. When a staggered cut is made in a sequence like this,
the overhangs are complementary (Figure 10.5).

FIGURE 10.5 In this (a) six-nucleotide restriction enzyme recognition site, notice that the sequence of six nucleotides reads the same in the
5' to 3' direction on one strand as it does in the 5' to 3' direction on the complementary strand. This is known as a palindrome. (b) The
restriction enzyme makes breaks in the DNA strands, and (c) the cut in the DNA results in “sticky ends”. Another piece of DNA cut on either
end by the same restriction enzyme could attach to these sticky ends and be inserted into the gap made by this cut.

Because these overhangs are capable of coming back together by hydrogen bonding with complementary overhangs
on a piece of DNA cut with the same restriction enzyme, these are called “sticky ends.” The process of forming
hydrogen bonds between complementary sequences on single strands to form double-stranded DNA is called
annealing. Addition of an enzyme called DNA ligase, which takes part in DNA replication in cells, permanently joins
the DNA fragments when the sticky ends come together. In this way, any DNA fragment can be spliced between the
two ends of a plasmid DNA that has been cut with the same restriction enzyme (Figure 10.6).
228 10 • Biotechnology

FIGURE 10.6 This diagram shows the steps involved in molecular cloning.

Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they contain new
combinations of genetic material. Proteins that are produced from recombinant DNA molecules are called
recombinant proteins. Not all recombinant plasmids are capable of expressing genes. Plasmids may also be
engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control
the expression of the recombinant proteins.

Reproductive Cloning
Reproductive cloning is a method used to make a clone or an identical copy of an entire multicellular organism.
Most multicellular organisms undergo reproduction by sexual means, which involves the contribution of DNA from
two individuals (parents), making it impossible to generate an identical copy or a clone of either parent. Recent

Access for free at openstax.org

 10.1 • Cloning and Genetic Engineering 229

advances in biotechnology have made it possible to reproductively clone mammals in the laboratory.

Natural sexual reproduction involves the union, during fertilization, of a sperm and an egg. Each of these gametes is
haploid, meaning they contain one set of chromosomes in their nuclei. The resulting cell, or zygote, is then diploid
and contains two sets of chromosomes. This cell divides mitotically to produce a multicellular organism. However,
the union of just any two cells cannot produce a viable zygote; there are components in the cytoplasm of the egg cell
that are essential for the early development of the embryo during its first few cell divisions. Without these
provisions, there would be no subsequent development. Therefore, to produce a new individual, both a diploid
genetic complement and an egg cytoplasm are required. The approach to producing an artificially cloned individual
is to take the egg cell of one individual and to remove the haploid nucleus. Then a diploid nucleus from a body cell of
a second individual, the donor, is put into the egg cell. The egg is then stimulated to divide so that development
proceeds. This sounds simple, but in fact it takes many attempts before each of the steps is completed successfully.

The first cloned agricultural animal was Dolly, a sheep who was born in 1996. The success rate of reproductive
cloning at the time was very low. Dolly lived for six years and died of a lung tumor (Figure 10.7). There was
speculation that because the cell DNA that gave rise to Dolly came from an older individual, the age of the DNA may
have affected her life expectancy. Since Dolly, several species of animals (such as horses, bulls, and goats) have
been successfully cloned.

There have been attempts at producing cloned human embryos as sources of embryonic stem cells. In the
procedure, the DNA from an adult human is introduced into a human egg cell, which is then stimulated to divide. The
technology is similar to the technology that was used to produce Dolly, but the embryo is never implanted into a
surrogate carrier. The cells produced are called embryonic stem cells because they have the capacity to develop into
many different kinds of cells, such as muscle or nerve cells. The stem cells could be used to research and ultimately
provide therapeutic applications, such as replacing damaged tissues. The benefit of cloning in this instance is that
the cells used to regenerate new tissues would be a perfect match to the donor of the original DNA. For example, a
leukemia patient would not require a sibling with a tissue match for a bone-marrow transplant. Freda Miller and
Elaine Fuchs, working independently, discovered stem cells in different layers of the skin. These cells help the skin
repair itself, and their discovery may have applications in treatments of skin disease and potentially other
conditions, such as nerve damage.
230 10 • Biotechnology

VISUAL CONNECTION

FIGURE 10.7 Dolly the sheep was the first agricultural animal to be cloned. To create Dolly, the nucleus was removed from a donor egg cell.
The enucleated egg was placed next to the other cell, then they were shocked to fuse. They were shocked again to start division. The cells
were allowed to divide for several days until an early embryonic stage was reached, before being implanted in a surrogate mother.

Why was Dolly a Finn-Dorset and not a Scottish Blackface sheep?

Genetic Engineering
Using recombinant DNA technology to modify an organism’s DNA to achieve desirable traits is called genetic
engineering. Addition of foreign DNA in the form of recombinant DNA vectors that are generated by molecular
cloning is the most common method of genetic engineering. An organism that receives the recombinant DNA is
called a genetically modified organism (GMO). If the foreign DNA that is introduced comes from a different species,
the host organism is called transgenic. Bacteria, plants, and animals have been genetically modified since the early
1970s for academic, medical, agricultural, and industrial purposes. These applications will be examined in more
detail in the next module.

LINK TO LEARNING
Watch this short video (http://openstax.org/l/transgenic) explaining how scientists create a transgenic animal.

Although the classic methods of studying the function of genes began with a given phenotype and determined the
genetic basis of that phenotype, modern techniques allow researchers to start at the DNA sequence level and ask:
"What does this gene or DNA element do?" This technique, called reverse genetics, has resulted in reversing the
classical genetic methodology. One example of this method is analogous to damaging a body part to determine its
function. An insect that loses a wing cannot fly, which means that the wing’s function is flight. The classic genetic
method compares insects that cannot fly with insects that can fly, and observes that the non-flying insects have lost
wings. Similarly in a reverse genetics approach, mutating or deleting genes provides researchers with clues about
gene function. Alternately, reverse genetics can be used to cause a gene to overexpress itself to determine what
phenotypic effects may occur.

Access for free at openstax.org

 10.2 • Biotechnology in Medicine and Agriculture 231

10.2 Biotechnology in Medicine and Agriculture

LEARNING OBJECTIVES
By the end of this section, you will be able to:
• Describe uses of biotechnology in medicine
• Describe uses of biotechnology in agriculture

It is easy to see how biotechnology can be used for medicinal purposes. Knowledge of the genetic makeup of our
species, the genetic basis of heritable diseases, and the invention of technology to manipulate and fix mutant genes
provides methods to treat diseases. Biotechnology in agriculture can enhance resistance to disease, pests, and
environmental stress to improve both crop yield and quality.

Genetic Diagnosis and Gene Therapy

The process of testing for suspected genetic defects before administering treatment is called genetic diagnosis by
genetic testing. In some cases in which a genetic disease is present in an individual’s family, family members may
be advised to undergo genetic testing. For example, mutations in the BRCA genes may increase the likelihood of
developing breast, ovarian, and some other cancers. A person with breast cancer can be screened for these
mutations. If one of the high-risk mutations is found, relatives may also wish to be screened for that particular
mutation, or simply be more vigilant for the occurrence of cancers. Genetic testing is also offered for fetuses (or
embryos with in vitro fertilization) to determine the presence or absence of disease-causing genes in families with
specific debilitating diseases.

LINK TO LEARNING
See how human DNA is extracted (http://openstax.org/l/DNA_extraction) for uses such as genetic testing.

Gene therapy is a genetic engineering technique that may one day be used to cure certain genetic diseases. In its
simplest form, it involves the introduction of a non-mutated gene at a random location in the genome to cure a
disease by replacing a protein that may be absent in these individuals because of a genetic mutation. The non-
mutated gene is usually introduced into diseased cells as part of a vector transmitted by a virus, such as an
adenovirus, that can infect the host cell and deliver the foreign DNA into the genome of the targeted cell (Figure
10.8). To date, gene therapies have been primarily experimental procedures in humans. A few of these experimental
treatments have been successful, but the methods may be important in the future as the factors limiting its success
are resolved.
232 10 • Biotechnology

FIGURE 10.8 This diagram shows the steps involved in curing disease with gene therapy using an adenovirus vector. (credit: modification of
work by NIH)

Production of Vaccines, Antibiotics, and Hormones

Traditional vaccination strategies use weakened or inactive forms of microorganisms or viruses to stimulate the
immune system. Modern techniques use specific genes of microorganisms cloned into vectors and mass-produced
in bacteria to make large quantities of specific substances to stimulate the immune system. The substance is then
used as a vaccine. In some cases, such as the H1N1 flu vaccine, genes cloned from the virus have been used to
combat the constantly changing strains of this virus.

Antibiotics kill bacteria and are naturally produced by microorganisms such as fungi; penicillin is perhaps the most
well-known example. Antibiotics are produced on a large scale by cultivating and manipulating fungal cells. The
fungal cells have typically been genetically modified to improve the yields of the antibiotic compound.

Recombinant DNA technology was used to produce large-scale quantities of the human hormone insulin in E. coli as
early as 1978. Previously, it was only possible to treat diabetes with pig insulin, which caused allergic reactions in
many humans because of differences in the insulin molecule. In addition, human growth hormone (HGH) is used to
treat growth disorders in children. The HGH gene was cloned from a cDNA (complementary DNA) library and
inserted into E. coli cells by cloning it into a bacterial vector.

Transgenic Animals
Although several recombinant proteins used in medicine are successfully produced in bacteria, some proteins need
a eukaryotic animal host for proper processing. For this reason, genes have been cloned and expressed in animals
such as sheep, goats, chickens, and mice. Animals that have been modified to express recombinant DNA are called
transgenic animals (Figure 10.9).

Access for free at openstax.org

 10.2 • Biotechnology in Medicine and Agriculture 233

FIGURE 10.9 It can be seen that two of these mice are transgenic because they have a gene that causes them to fluoresce under a UV light.
The non-transgenic mouse does not have the gene that causes fluorescence. (credit: Ingrid Moen et al.)

Several human proteins are expressed in the milk of transgenic sheep and goats. In one commercial example, the
FDA has approved a blood anticoagulant protein that is produced in the milk of transgenic goats for use in humans.
Mice have been used extensively for expressing and studying the effects of recombinant genes and mutations.

Transgenic Plants
Manipulating the DNA of plants (creating genetically modified organisms, or GMOs) has helped to create desirable
traits such as disease resistance, herbicide, and pest resistance, better nutritional value, and better shelf life (Figure
10.10). Plants are the most important source of food for the human population. Farmers developed ways to select
for plant varieties with desirable traits long before modern-day biotechnology practices were established.

FIGURE 10.10 Corn, a major agricultural crop used to create products for a variety of industries, is often modified through plant
biotechnology. (credit: Keith Weller, USDA)

Transgenic plants have received DNA from other species. Because they contain unique combinations of genes and
are not restricted to the laboratory, transgenic plants and other GMOs are closely monitored by government
agencies to ensure that they are fit for human consumption and do not endanger other plant and animal life.
234 10 • Biotechnology

Because foreign genes can spread to other species in the environment, particularly in the pollen and seeds of plants,
extensive testing is required to ensure ecological stability. Staples like corn, potatoes, and tomatoes were the first
crop plants to be genetically engineered.

Transformation of Plants Using Agrobacterium tumefaciens

In plants, tumors caused by the bacterium Agrobacterium tumefaciens occur by transfer of DNA from the bacterium
to the plant. The artificial introduction of DNA into plant cells is more challenging than in animal cells because of the
thick plant cell wall. Researchers used the natural transfer of DNA from Agrobacterium to a plant host to introduce
DNA fragments of their choice into plant hosts. In nature, the disease-causing A. tumefaciens have a set of plasmids
that contain genes that integrate into the infected plant cell’s genome. Researchers manipulate the plasmids to
carry the desired DNA fragment and insert it into the plant genome.

The Organic Insecticide Bacillus thuringiensis

Bacillus thuringiensis (Bt) is a bacterium that produces protein crystals that are toxic to many insect species that
feed on plants. Insects that have eaten Bt toxin stop feeding on the plants within a few hours. After the toxin is
activated in the intestines of the insects, death occurs within a couple of days. The crystal toxin genes have been
cloned from the bacterium and introduced into plants, therefore allowing plants to produce their own crystal Bt
toxin that acts against insects. Bt toxin is safe for the environment and non-toxic to mammals (including humans).
As a result, it has been approved for use by organic farmers as a natural insecticide. There is some concern,
however, that insects may evolve resistance to the Bt toxin in the same way that bacteria evolve resistance to
antibiotics.

FlavrSavr Tomato
The first GM crop to be introduced into the market was the FlavrSavr Tomato produced in 1994. Molecular genetic
technology was used to slow down the process of softening and rotting caused by fungal infections, which led to
increased shelf life of the GM tomatoes. Additional genetic modification improved the flavor of this tomato. The
FlavrSavr tomato did not successfully stay in the market because of problems maintaining and shipping the crop.

10.3 Genomics and Proteomics

LEARNING OBJECTIVES
By the end of this section, you will be able to:
• Define genomics and proteomics
• Define whole genome sequencing
• Explain different applications of genomics and proteomics

The study of nucleic acids began with the discovery of DNA, progressed to the study of genes and small fragments,
and has now exploded to the field of genomics. Genomics is the study of entire genomes, including the complete set
of genes, their nucleotide sequence and organization, and their interactions within a species and with other species.
The advances in genomics have been made possible by DNA sequencing technology. Just as information technology
has led to Google Maps that enable us to get detailed information about locations around the globe, genomic
information is used to create similar maps of the DNA of different organisms.

Mapping Genomes
Genome mapping is the process of finding the location of genes on each chromosome. The maps that are created
are comparable to the maps that we use to navigate streets. A genetic map is an illustration that lists genes and
their location on a chromosome. Genetic maps provide the big picture (similar to a map of interstate highways) and
use genetic markers (similar to landmarks). A genetic marker is a gene or sequence on a chromosome that shows
genetic linkage with a trait of interest. The genetic marker tends to be inherited with the gene of interest, and one
measure of distance between them is the recombination frequency during meiosis. Early geneticists called this
linkage analysis.

Physical maps get into the intimate details of smaller regions of the chromosomes (similar to a detailed road map)
(Figure 10.11). A physical map is a representation of the physical distance, in nucleotides, between genes or genetic
markers. Both genetic linkage maps and physical maps are required to build a complete picture of the genome.
Having a complete map of the genome makes it easier for researchers to study individual genes. Human genome

Access for free at openstax.org

 10.3 • Genomics and Proteomics 235

maps help researchers in their efforts to identify human disease-causing genes related to illnesses such as cancer,
heart disease, and cystic fibrosis, to name a few. In addition, genome mapping can be used to help identify
organisms with beneficial traits, such as microbes with the ability to clean up pollutants or even prevent pollution.
Research involving plant genome mapping may lead to methods that produce higher crop yields or to the
development of plants that adapt better to climate change.

FIGURE 10.11 This is a physical map of the human X chromosome. (credit: modification of work by NCBI, NIH)

Genetic maps provide the outline, and physical maps provide the details. It is easy to understand why both types of
genome-mapping techniques are important to show the big picture. Information obtained from each technique is
used in combination to study the genome. Genomic mapping is used with different model organisms that are used
for research. Genome mapping is still an ongoing process, and as more advanced techniques are developed, more
advances are expected. Genome mapping is similar to completing a complicated puzzle using every piece of
available data. Mapping information generated in laboratories all over the world is entered into central databases,
such as the National Center for Biotechnology Information (NCBI). Efforts are made to make the information more
easily accessible to researchers and the general public. Just as we use global positioning systems instead of paper
maps to navigate through roadways, NCBI allows us to use a genome viewer tool to simplify the data mining
process.
236 10 • Biotechnology

LINK TO LEARNING
Online Mendelian Inheritance in Man (OMIM) (http://openstax.org/l/OMIM2) is a searchable online catalog of human
genes and genetic disorders. This website shows genome mapping, and also details the history and research of each
trait and disorder. Click the link to search for traits (such as handedness) and genetic disorders (such as diabetes).

Whole Genome Sequencing

Although there have been significant advances in the medical sciences in recent years, doctors are still confounded
by many diseases and researchers are using whole genome sequencing to get to the bottom of the problem. Whole
genome sequencing is a process that determines the DNA sequence of an entire genome. Whole genome
sequencing is a brute-force approach to problem solving when there is a genetic basis at the core of a disease.
Several laboratories now provide services to sequence, analyze, and interpret entire genomes.

In 2010, whole genome sequencing was used to save a young boy whose intestines had multiple mysterious
abscesses. The child had several colon operations with no relief. Finally, a whole genome sequence revealed a
defect in a pathway that controls apoptosis (programmed cell death). A bone marrow transplant was used to
overcome this genetic disorder, leading to a cure for the boy. He was the first person to be successfully diagnosed
using whole genome sequencing.

The first genomes to be sequenced, such as those belonging to viruses, bacteria, and yeast, were smaller in terms of
the number of nucleotides than the genomes of multicellular organisms. The genomes of other model organisms,
such as the mouse (Mus musculus), the fruit fly (Drosophila melanogaster), and the nematode (Caenorhabditis
elegans) are now known. A great deal of basic research is performed in model organisms because the information
can be applied to other organisms. A model organism is a species that is studied as a model to understand the
biological processes in other species that can be represented by the model organism. For example, fruit flies are
able to metabolize alcohol like humans, so the genes affecting sensitivity to alcohol have been studied in fruit flies in
an effort to understand the variation in sensitivity to alcohol in humans. Having entire genomes sequenced helps
with the research efforts in these model organisms (Figure 10.12).

FIGURE 10.12 Much basic research is done with model organisms, such as the mouse, Mus musculus; the fruit fly, Drosophila
melanogaster; the nematode Caenorhabditis elegans; the yeast Saccharomyces cerevisiae; and the common weed, Arabidopsis thaliana.
(credit "mouse": modification of work by Florean Fortescue; credit "nematodes": modification of work by "snickclunk"/Flickr; credit
"common weed": modification of work by Peggy Greb, USDA; scale-bar data from Matt Russell)

The first human genome sequence was published in 2003. The number of whole genomes that have been
sequenced steadily increases and now includes hundreds of species and thousands of individual human genomes.

Access for free at openstax.org

 10.3 • Genomics and Proteomics 237

Applying Genomics
The introduction of DNA sequencing and whole genome sequencing projects, particularly the Human Genome
Project, has expanded the applicability of DNA sequence information. Genomics is now being used in a wide variety
of fields, such as metagenomics, pharmacogenomics, and mitochondrial genomics. The most commonly known
application of genomics is to understand and find cures for diseases.

Predicting Disease Risk at the Individual Level

Predicting the risk of disease involves screening and identifying currently healthy individuals by genome analysis at
the individual level. Intervention with lifestyle changes and drugs can be recommended before disease onset.
However, this approach is most applicable when the problem arises from a single gene mutation. Such defects only
account for about 5 percent of diseases found in developed countries. Most of the common diseases, such as heart
disease, are multifactorial or polygenic, which refers to a phenotypic characteristic that is determined by two or
more genes, and also environmental factors such as diet. In April 2010, scientists at Stanford University published
the genome analysis of a healthy individual (Stephen Quake, a scientist at Stanford University, who had his genome
sequenced); the analysis predicted his propensity to acquire various diseases. A risk assessment was done to
analyze Quake’s percentage of risk for 55 different medical conditions. A rare genetic mutation was found that
showed him to be at risk for sudden heart attack. He was also predicted to have a 23 percent risk of developing
prostate cancer and a 1.4 percent risk of developing Alzheimer’s disease. The scientists used databases and several
publications to analyze the genomic data. Even though genomic sequencing is becoming more affordable and
analytical tools are becoming more reliable, ethical issues surrounding genomic analysis at a population level
remain to be addressed. For example, could such data be legitimately used to charge more or less for insurance or
to affect credit ratings?

Gene Editing
For thousands of years, humans have engaged in some level of control over genes and heredity regarding the plants
and animals we rely on. The technology now exists to exert that control more directly by precisely altering the DNA
of organisms. The technique is usually referred to as CRISPR, for the portions of DNA it targets: "Clustered Regularly
Interspaced Short Palindromic Repeats." In essence, DNA contains repetitive sequences with "spacers" between
them. CRISPR-associated nucleases (known as "Cas") are enzymes that can identify, attach to, and cut the strand at
precise locations. In 2012, Jennifer Doudna and Emmanuelle Charpentier developed a method to combine the Cas
nuclease with a synthetically produced "guide RNA" that leads the nuclease to selected locations on the DNA
strand. The discovery revolutionized gene editing. Researchers around the world have used CRISPR to manipulate
the actual DNA of plants, animals, laboratory cell lines, and (in trials) human patients. Doudna and Charpentier were
awarded the Nobel Prize for their work.

Gene editing is so promising because it can be used experimentally to understand disease or organismal limitations,
then be applied to overcome those issues. For example, in a human trial, cancerous cells were removed from a
person, edited to remove their cancerous properties at the DNA level, and reintroduced into the patient so that
those now edited cells could multiply and replace the cancerous ones. Using the person's own cells increases the
likelihood of acceptance and success.

Like other applications of genomics, the prospect of directly editing genes brings up a number of ethical issues. Both
Doudna and Chapentier, as well as many other genetic engineers, support only certain CRISPR applications. And
many governments and other entities place strict guidelines on the uses of the powerful technology.

Genome-wide Association Studies

Since 2005, it has been possible to conduct a type of study called a genome-wide association study, or GWAS. A
GWAS is a method that identifies differences between individuals in single nucleotide polymorphisms (SNPs) that
may be involved in causing diseases. The method is particularly suited to diseases that may be affected by one or
many genetic changes throughout the genome. It is very difficult to identify the genes involved in such a disease
using family history information. The GWAS method relies on a genetic database that has been in development since
2002 called the International HapMap Project. The HapMap Project sequenced the genomes of several hundred
individuals from around the world and identified groups of SNPs. The groups include SNPs that are located near to
each other on chromosomes so they tend to stay together through recombination. The fact that the group stays
together means that identifying one marker SNP is all that is needed to identify all the SNPs in the group. There are
238 10 • Biotechnology

several million SNPs identified, but identifying them in other individuals who have not had their complete genome
sequenced is much easier because only the marker SNPs need to be identified.

In a common design for a GWAS, two groups of individuals are chosen; one group has the disease, and the other
group does not. The individuals in each group are matched in other characteristics to reduce the effect of
confounding variables causing differences between the two groups. For example, the genotypes may differ because
the two groups are mostly taken from different parts of the world. Once the individuals are chosen, and typically
their numbers are a thousand or more for the study to work, samples of their DNA are obtained. The DNA is analyzed
using automated systems to identify large differences in the percentage of particular SNPs between the two groups.
Often the study examines a million or more SNPs in the DNA. The results of GWAS can be used in two ways: the
genetic differences may be used as markers for susceptibility to the disease in undiagnosed individuals, and the
particular genes identified can be targets for research into the molecular pathway of the disease and potential
therapies. An offshoot of the discovery of gene associations with disease has been the formation of companies that
provide so-called “personal genomics” that will identify risk levels for various diseases based on an individual’s SNP
complement. The science behind these services is controversial.

Because GWAS looks for associations between genes and disease, these studies provide data for other research into
causes, rather than answering specific questions themselves. An association between a gene difference and a
disease does not necessarily mean there is a cause-and-effect relationship. However, some studies have provided
useful information about the genetic causes of diseases. For example, three different studies in 2005 identified a
gene for a protein involved in regulating inflammation in the body that is associated with a disease-causing
blindness called age-related macular degeneration. This opened up new possibilities for research into the cause of
this disease. A large number of genes have been identified to be associated with Crohn’s disease using GWAS, and
some of these have suggested new hypothetical mechanisms for the cause of the disease.

Pharmacogenomics
Pharmacogenomics involves evaluating the effectiveness and safety of drugs on the basis of information from an
individual's genomic sequence. Personal genome sequence information can be used to prescribe medications that
will be most effective and least toxic on the basis of the individual patient’s genotype. Studying changes in gene
expression could provide information about the gene transcription profile in the presence of the drug, which can be
used as an early indicator of the potential for toxic effects. For example, genes involved in cellular growth and
controlled cell death, when disturbed, could lead to the growth of cancerous cells. Genome-wide studies can also
help to find new genes involved in drug toxicity. The gene signatures may not be completely accurate, but can be
tested further before pathologic symptoms arise.

Metagenomics
Traditionally, microbiology has been taught with the view that microorganisms are best studied under pure culture
conditions, which involves isolating a single type of cell and culturing it in the laboratory. Because microorganisms
can go through several generations in a matter of hours, their gene expression profiles adapt to the new laboratory
environment very quickly. On the other hand, many species resist being cultured in isolation. Most microorganisms
do not live as isolated entities, but in microbial communities known as biofilms. For all of these reasons, pure
culture is not always the best way to study microorganisms. Metagenomics is the study of the collective genomes of
multiple species that grow and interact in an environmental niche. Metagenomics can be used to identify new
species more rapidly and to analyze the effect of pollutants on the environment (Figure 10.13). Metagenomics
techniques can now also be applied to communities of higher eukaryotes, such as fish.

Access for free at openstax.org

 10.3 • Genomics and Proteomics 239

FIGURE 10.13 Metagenomics involves isolating DNA from multiple species within an environmental niche. The DNA is cut up and
sequenced, allowing entire genome sequences of multiple species to be reconstructed from the sequences of overlapping pieces.

Creation of New Biofuels

Knowledge of the genomics of microorganisms is being used to find better ways to harness biofuels from algae and
cyanobacteria. The primary sources of fuel today are coal, oil, wood, and other plant products such as ethanol.
Although plants are renewable resources, there is still a need to find more alternative renewable sources of energy
to meet our population’s energy demands. The microbial world is one of the largest resources for genes that encode
new enzymes and produce new organic compounds, and it remains largely untapped. This vast genetic resource
holds the potential to provide new sources of biofuels (Figure 10.14).

FIGURE 10.14 Renewable fuels were tested in Navy ships and aircraft at the first Naval Energy Forum. (credit: modification of work by John
F. Williams, US Navy)
240 10 • Biotechnology

Mitochondrial Genomics
Mitochondria are intracellular organelles that contain their own DNA. Mitochondrial DNA mutates at a rapid rate and
is often used to study evolutionary relationships. Another feature that makes studying the mitochondrial genome
interesting is that in most multicellular organisms, the mitochondrial DNA is passed on from the mother during the
process of fertilization. For this reason, mitochondrial genomics is often used to trace genealogy.

Genomics in Forensic Analysis

Information and clues obtained from DNA samples found at crime scenes have been used as evidence in court
cases, and genetic markers have been used in forensic analysis. Genomic analysis has also become useful in this
field. In 2001, the first use of genomics in forensics was published. It was a collaborative effort between academic
research institutions and the FBI to solve the mysterious cases of anthrax (Figure 10.15) that was transported by
the US Postal Service. Anthrax bacteria were made into an infectious powder and mailed to news media and two
U.S. Senators. The powder infected the administrative staff and postal workers who opened or handled the letters.
Five people died, and 17 were sickened from the bacteria. Using microbial genomics, researchers determined that a
specific strain of anthrax was used in all the mailings; eventually, the source was traced to a scientist at a national
biodefense laboratory in Maryland.

FIGURE 10.15 Bacillus anthracis is the organism that causes anthrax. (credit: modification of work by CDC; scale-bar data from Matt
Russell)

Genomics in Agriculture
Genomics can reduce the trials and failures involved in scientific research to a certain extent, which could improve
the quality and quantity of crop yields in agriculture (Figure 10.16). Linking traits to genes or gene signatures helps
to improve crop breeding to generate hybrids with the most desirable qualities. Scientists use genomic data to
identify desirable traits, and then transfer those traits to a different organism to create a new genetically modified
organism, as described in the previous module. Scientists are discovering how genomics can improve the quality
and quantity of agricultural production. For example, scientists could use desirable traits to create a useful product
or enhance an existing product, such as making a drought-sensitive crop more tolerant of the dry season.

Access for free at openstax.org

 10.3 • Genomics and Proteomics 241

FIGURE 10.16 Transgenic agricultural plants can be made to resist disease. These transgenic plums are resistant to the plum pox virus.
(credit: Scott Bauer, USDA ARS)

Proteomics
Proteins are the final products of genes that perform the function encoded by the gene. Proteins are composed of
amino acids and play important roles in the cell. All enzymes (except ribozymes) are proteins and act as catalysts
that affect the rate of reactions. Proteins are also regulatory molecules, and some are hormones. Transport proteins,
such as hemoglobin, help transport oxygen to various organs. Antibodies that defend against foreign particles are
also proteins. In the diseased state, protein function can be impaired because of changes at the genetic level or
because of direct impact on a specific protein.

A proteome is the entire set of proteins produced by a cell type. Proteomes can be studied using the knowledge of
genomes because genes code for mRNAs, and the mRNAs encode proteins. The study of the function of proteomes
is called proteomics. Proteomics complements genomics and is useful when scientists want to test their
hypotheses that were based on genes. Even though all cells in a multicellular organism have the same set of genes,
the set of proteins produced in different tissues is different and dependent on gene expression. Thus, the genome is
constant, but the proteome varies and is dynamic within an organism. In addition, RNAs can be alternatively spliced
(cut and pasted to create novel combinations and novel proteins), and many proteins are modified after translation.
Although the genome provides a blueprint, the final architecture depends on several factors that can change the
progression of events that generate the proteome.

Genomes and proteomes of patients suffering from specific diseases are being studied to understand the genetic
basis of the disease. The most prominent disease being studied with proteomic approaches is cancer (Figure 10.17).
Proteomic approaches are being used to improve the screening and early detection of cancer; this is achieved by
identifying proteins whose expression is affected by the disease process. An individual protein is called a
biomarker, whereas a set of proteins with altered expression levels is called a protein signature. For a biomarker or
protein signature to be useful as a candidate for early screening and detection of a cancer, it must be secreted in
body fluids such as sweat, blood, or urine, so that large-scale screenings can be performed in a noninvasive fashion.
The current problem with using biomarkers for the early detection of cancer is the high rate of false-negative results.
A false-negative result is a negative test result that should have been positive. In other words, many cases of cancer
go undetected, which makes biomarkers unreliable. Some examples of protein biomarkers used in cancer detection
are CA-125 for ovarian cancer and PSA for prostate cancer. Protein signatures may be more reliable than biomarkers
to detect cancer cells. Proteomics is also being used to develop individualized treatment plans, which involves the
prediction of whether or not an individual will respond to specific drugs and the side effects that the individual may
have. Proteomics is also being used to predict the possibility of disease recurrence.
242 10 • Biotechnology

FIGURE 10.17 This machine is preparing to do a proteomic pattern analysis to identify specific cancers so that an accurate cancer
prognosis can be made. (credit: Dorie Hightower, NCI, NIH)

The National Cancer Institute has developed programs to improve the detection and treatment of cancer. The
Clinical Proteomic Technologies for Cancer and the Early Detection Research Network are efforts to identify protein
signatures specific to different types of cancers. The Biomedical Proteomics Program is designed to identify protein
signatures and design effective therapies for cancer patients.

Access for free at openstax.org

 10 • Key Terms 243

Key Terms
anneal in molecular biology, the process by which a model to understand the biological processes in
two single strands of DNA hydrogen bond at other species represented by the model organism
complementary nucleotides to form a double- pharmacogenomics the study of drug interactions
stranded molecule with the genome or proteome; also called
biomarker an individual protein that is uniquely toxicogenomics
produced in a diseased state physical map a representation of the physical
biotechnology the use of artificial methods to modify distance between genes or genetic markers
the genetic material of living organisms or cells to plasmid a small circular molecule of DNA found in
produce novel compounds or to perform new bacteria that replicates independently of the main
functions bacterial chromosome; plasmids code for some
cloning the production of an exact copy—specifically, important traits for bacteria and can be used as
an exact genetic copy—of a gene, cell, or organism vectors to transport DNA into bacteria in genetic
gel electrophoresis a technique used to separate engineering applications
molecules on the basis of their ability to migrate polymerase chain reaction (PCR) a technique used
through a semisolid gel in response to an electric to make multiple copies of DNA
current protein signature a set of over- or under-expressed
gene therapy the technique used to cure heritable proteins characteristic of cells in a particular
diseases by replacing mutant genes with good diseased tissue
genes proteomics study of the function of proteomes
genetic engineering alteration of the genetic makeup recombinant DNA a combination of DNA fragments
of an organism using the molecular methods of generated by molecular cloning that does not exist
biotechnology in nature
genetic map an outline of genes and their location on recombinant protein a protein that is expressed from
a chromosome that is based on recombination recombinant DNA molecules
frequencies between markers reproductive cloning cloning of entire organisms
genetic testing identifying gene variants in an restriction enzyme an enzyme that recognizes a
individual that may lead to a genetic disease in that specific nucleotide sequence in DNA and cuts the
individual DNA double strand at that recognition site, often
genetically modified organism (GMO) an organism with a staggered cut leaving short single strands or
whose genome has been artificially changed “sticky” ends
genomics the study of entire genomes, including the reverse genetics a form of genetic analysis that
complete set of genes, their nucleotide sequence manipulates DNA to disrupt or affect the product of
and organization, and their interactions within a a gene to analyze the gene’s function
species and with other species transgenic describing an organism that receives DNA
metagenomics the study of the collective genomes of from a different species
multiple species that grow and interact in an whole genome sequencing a process that
environmental niche determines the nucleotide sequence of an entire
model organism a species that is studied and used as genome

Chapter Summary
10.1 Cloning and Genetic Engineering modifying them to achieve desirable traits.

Nucleic acids can be isolated from cells for the Cloning may involve cloning small DNA fragments
purposes of further analysis by breaking open the cells (molecular cloning), or cloning entire organisms
and enzymatically destroying all other major (reproductive cloning). In molecular cloning with
macromolecules. Fragmented or whole chromosomes bacteria, a desired DNA fragment is inserted into a
can be separated on the basis of size by gel bacterial plasmid using restriction enzymes and the
electrophoresis. Short stretches of DNA can be plasmid is taken up by a bacterium, which will then
amplified by PCR. DNA can be cut (and subsequently express the foreign DNA. Using other techniques,
re-spliced together) using restriction enzymes. The foreign genes can be inserted into eukaryotic
molecular and cellular techniques of biotechnology organisms. In each case, the organisms are called
allow researchers to genetically engineer organisms, transgenic organisms. In reproductive cloning, a donor
244 10 • Visual Connection Questions

nucleus is put into an enucleated egg cell, which is provide an outline for the location of genes within a
then stimulated to divide and develop into an genome, and they estimate the distance between
organism. genes and genetic markers on the basis of the
recombination frequency during meiosis. Physical
In reverse genetics methods, a gene is mutated or
maps provide detailed information about the physical
removed in some way to identify its effect on the
distance between the genes. The most detailed
phenotype of the whole organism as a way to
information is available through sequence mapping.
determine its function.
Information from all mapping and sequencing sources
10.2 Biotechnology in Medicine and is combined to study an entire genome.
Agriculture Whole genome sequencing is the latest available
Genetic testing is performed to identify disease- resource to treat genetic diseases. Some doctors are
causing genes, and can be used to benefit affected using whole genome sequencing to save lives.
individuals and their relatives who have not developed Genomics has many industrial applications, including
disease symptoms yet. Gene therapy—by which biofuel development, agriculture, pharmaceuticals, and
functioning genes are incorporated into the genomes of pollution control.
individuals with a non-functioning mutant gene—has Imagination is the only barrier to the applicability of
the potential to cure heritable diseases. Transgenic genomics. Genomics is being applied to most fields of
organisms possess DNA from a different species, biology; it can be used for personalized medicine,
usually generated by molecular cloning techniques. prediction of disease risks at an individual level, the
Vaccines, antibiotics, and hormones are examples of study of drug interactions before the conduction of
products obtained by recombinant DNA technology. clinical trials, and the study of microorganisms in the
Transgenic animals have been created for experimental environment as opposed to the laboratory. It is also
purposes and some are used to produce some human being applied to the generation of new biofuels,
proteins. genealogical assessment using mitochondria,
Genes are inserted into plants, using plasmids in the advances in forensic science, and improvements in
bacterium Agrobacterium tumefaciens, which infects agriculture.
plants. Transgenic plants have been created to improve Proteomics is the study of the entire set of proteins
the characteristics of crop plants—for example, by expressed by a given type of cell under certain
giving them insect resistance by inserting a gene for a environmental conditions. In a multicellular organism,
bacterial toxin. different cell types will have different proteomes, and
10.3 Genomics and Proteomics these will vary with changes in the environment. Unlike
a genome, a proteome is dynamic and under constant
Genome mapping is similar to solving a big, flux, which makes it more complicated and more useful
complicated puzzle with pieces of information coming than the knowledge of genomes alone.
from laboratories all over the world. Genetic maps

Visual Connection Questions

1. Figure 10.7 Why was Dolly a Finn-Dorset and not a
Scottish Blackface sheep?

Review Questions
2. In gel electrophoresis of DNA, the different bands in 3. In the reproductive cloning of an animal, the
the final gel form because the DNA molecules genome of the cloned individual comes from
________. ________.
a. are from different organisms a. a sperm cell
b. have different lengths b. an egg cell
c. have different nucleotide compositions c. any gamete cell
d. have different genes d. a body cell

Access for free at openstax.org

 10 • Critical Thinking Questions 245

4. What carries a gene from one organism into a 7. What is the most challenging issue facing genome
bacteria cell? sequencing?
a. a plasmid a. the inability to develop fast and accurate
b. an electrophoresis gel sequencing techniques
c. a restriction enzyme b. the ethics of using information from genomes
d. polymerase chain reaction at the individual level
c. the availability and stability of DNA
5. What is a genetically modified organism (GMO)? d. all of the above
a. a plant with certain genes removed
b. an organism with an artificially altered genome 8. Genomics can be used in agriculture to:
c. a hybrid organism a. generate new hybrid strains
d. any agricultural organism produced by b. improve disease resistance
breeding or biotechnology c. improve yield
d. all of the above
6. What is the role of Agrobacterium tumefaciens in
the production of transgenic plants? 9. What kind of diseases are studied using genome-
a. Genes from A. tumefaciens are inserted into wide association studies?
plant DNA to give the plant different traits. a. viral diseases
b. Transgenic plants have been given resistance b. single-gene inherited diseases
to the pest A. tumefaciens. c. diseases caused by multiple genes
c. A. tumefaciens is used as a vector to move d. diseases caused by environmental factors
genes into plant cells.
d. Plant genes are incorporated into the genome
of Agrobacterium tumefaciens.

Critical Thinking Questions

10. What is the purpose and benefit of the polymerase 12. Describe two of the applications for genome
chain reaction? mapping.

11. Today, it is possible for a diabetic patient to 13. Identify a possible advantage and a possible
purchase human insulin from a pharmacist. What disadvantage of a genetic test that would identify
technology makes this possible and why is it a genes in individuals that increase their probability
benefit over how things used to be? of having Alzheimer's disease later in life.