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CHAPTER 15
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Molecular and Genomic Surgery
1. The process that occurs during translational control of
eukaryotic gene expression is:
A. Protein degradation.
B. RNA processing.
C. Posttranslational control.
D. Transcription.
2. In the transcription of prokaryotes, binding of RNA
polymerase to the specific promoter region is achieved
by:
A. Sigma factors
B. Operon
C. Elongation factors
D. Rho factors
Answer: A
See Schwartz 11th ed., Figure 15-4, p. 483.
Answer: A
Initiation of transcription in prokaryotes (bacteria) begins
with the recognition of DNA sequences by RNA polymerase.
First, the bacterial RNA polymerase catalyzes RNA synthesis
through loose binding to any region in the double-stranded
DNA and then through specific binding to the promoter
region with the assistance of accessory proteins called σ factors (sigma factors). A promoter region is the DNA region
upstream of the transcription initiation site. RNA polymerase binds tightly at the promoter sites and causes the
double-stranded DNA structure to unwind. Consequently,
few nucleotides can be base-paired with the DNA template to
begin transcription. Once transcription begins, the σ factor
is released. The growing RNA chain may begin to peel off as
the chain elongates. This occurs in such a way that there are
always about 10 to 12 nucleotides of the growing RNA chains
that are base-paired with the DNA template. (See Schwartz
11th ed., Figure 15-4, p. 483.)
3. All of the following transcription mechanisms occur in
eukaryotes EXCEPT:
A. Chromatin structure changes to allow DNA to be
accessible to the polymerase.
B. Three separate RNA polymerases are involved.
C. Proteins or initiation factors are not required.
D. Often packaged with histone and nonhistone pro-
teins into chromatins.
Answer: C
The unique features of eukaryotic transcription are as follows:
(a) Three separate RNA polymerases are involved in eukaryotes: RNA polymerase I transcribes the precursor of 5.8S, 18S,
and 28S rRNAs; RNA polymerase II synthesizes the precursors of mRNA as well as microRNA; and RNA polymerase
III makes tRNAs and 5S rRNAs. (b) In eukaryotes, the initial
transcript is often the precursor to final mRNAs, tRNAs, and
rRNAs. The precursor is then modified and/or processed into
its final functional form. RNA splicing is one type of processing to remove the noncoding introns (the region between
coding exons) on an mRNA. (c) In contrast to bacterial DNA,
eukaryotic DNA often is packaged with histone and nonhistone proteins into chromatins. Transcription will only occur
when the chromatin structure changes in such a way that
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CHAPTER 15
4. Which of the following statements is incorrect?
Molecular and Genomic Surgery
A. A codon is a triplet of three bases that codes for a
single amino acid.
B. More than one triplet codes for the same amino acid.
C. Codons are mRNA sequentially recognized by tRNA
adaptor proteins.
D. Protein synthesis proceeds in the carboxy-to-amino-
terminus direction.
DNA is accessible to the polymerase. (d) RNA is made in
the nucleus and transported into cytoplasm, where translation occurs. Therefore, unlike bacteria, eukaryotes undergo
uncoupled transcription and translation. (See Schwartz 11th
ed., Figure 15-4, p. 483.)
Answer: D
A codon, a triplet of three bases, codes for one amino acid. In
this case, random combinations of the four bases form 4 ×
4 × 4, or 64 codes. Because 64 codes are more than enough
for 20 amino acids, most amino acids are coded by more than
one codon. The start codon is AUG, which also corresponds
to methionine; therefore, almost all proteins begin with this
amino acid. The sequence of nucleotide triplets that follows
the start codon signal is termed the reading frame. The codons
on mRNA are sequentially recognized by tRNA adaptor proteins. Specific enzymes termed aminoacyl-tRNA synthetases
link a specific amino acid to a specific tRNA. The translation
of mRNA to protein requires the ribosomal complex to move
stepwise along the mRNA until the initiator methionine
sequence is identified. In concert with various protein initiator factors, the methionyl-tRNA is positioned on the mRNA
and protein synthesis begins. Each new amino acid is added
sequentially by the appropriate tRNA in conjunction with
proteins called elongation factors. Protein synthesis proceeds
in the amino-to-carboxy-terminus direction. (See Schwartz
11th ed., p. 484.)
5. The process of decoding information on mRNA to synthesize proteins is called:
A. Transcription.
B. Translation.
C. Replication.
D. Signaling.
DNA
Transcription
RNA
transcript
Transcriptional
control
Nuclear envelope
RNA
processing
Posttranscriptional
control
Nucleus
mRNA mRNA Protein
Cytoplasm
degradation
RNA
transport
Answer: B
DNA directs the synthesis of RNA; RNA in turn directs the
synthesis of proteins. Proteins are variable-length polypeptide polymers composed of various combinations of 20 different amino acids and are the working molecules of the cell.
The process of decoding information on mRNA to synthesize
proteins is called translation (see Fig. 15-1). Translation takes
place in ribosomes composed of rRNA and ribosomal proteins. (See Schwartz 11th ed., p. 483.)
mRNA
turnover
RNA
Translation
Translational
control
Protein
turnover
Protein
degradation
Posttranslational
modification
Posttranslational
control
Active
protein
FIG. 15-1. Four major steps in the control of eukaryotic gene expression. Transcriptional and posttranscriptional control determine the level
of messenger RNA (mRNA) that is available to make a protein, while translational and posttranslational control determine the final outcome
of functional proteins. Note that posttranscriptional and posttranslational controls consist of several steps.
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B/CDK1
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6. The human genome contains approximately:
A. 35,000 to 40,000 genes.
B. 20,000 to 25,000 genes.
C. 25,000 to 30,000 genes.
D. 30,000 to 35,000 genes.
7. If chronic kidney disease (CKD) is to a cell as an engine
is to a car, then cyclins and cyclin-dependent kinase
inhibitors.(CKI) are:
A. The key and ignition, respectively.
B. The gas pedal and brakes, respectively.
C. The distributor and the spark plug, respectively.
D. The windows and the tires, respectively.
Answer: C
The human genome has an estimated 25,000 to 30,000 genes,
and overall it is 99.9% identical in all people. Approximately
3 million locations where single-base DNA differences exist have
been identified and termed single nucleotide polymorphisms.
Single nucleotide polymorphisms may be critical determinants
of human variation in disease susceptibility and responses to
environmental factors. (See Schwartz 11th ed., p. 485.)
Answer: B
The cell cycle is connected with signal transduction pathways
as well as gene expression. Although the S and M phases rarely
are subjected to changes imposed by extracellular signals, the
G1 and G2 phases are the primary periods when cells decide
whether to move on to the next phase. During the G1 phase,
cells receive green- or red-light signals, S phase entry or G1
arrest, respectively. Growing cells proliferate only when supplied with appropriate mitogenic growth factors. Cells become
committed to entry of the cell cycle only toward the end of
G1. Mitogenic signals stimulate the activity of early G1 CDKs
(eg, cyclin D/CDK4) that inhibit the activity of pRb protein
and activate the transcription factor called E2F to induce the
expression of batteries of genes essential for G1-S progression.
Meanwhile, cells also receive antiproliferative signals such as
those from tumor suppressors. These antiproliferative signals
also act in the G1 phase to stop cells’ progress into the S phase
by inducing CKI production. For example, when DNA is damaged, cells will repair the damage before entering the S phase.
Therefore, G1 contains one of the most important checkpoints
for cell cycle progression. If the analogy is made that CDK is
to a cell as an engine is to a car, then cyclins and CKI are the
gas pedal and brake, respectively. Accelerated proliferation or
improper cell cycle progression with damaged DNA would be
disastrous. Genetic gain-of-function mutations in oncogenes
(that often promote expression or activity of the cyclin/CDK
complex) or loss-of-function mutations in tumor suppressor (that stimulate production of CKI) are causal factors for
malignant transformation. (See Schwartz 11th ed., p. 486.)
CHAPTER 15
Molecular and Genomic Surgery
8. The cell cycle period during which DNA is replicated is:
A. S.
B. G1.
C. M.
D. G2.
FIG. 15-2. The cell cycle and its control system. M is the mitosis
phase, when the nucleus and the cytoplasm divide; S is the phase
when DNA is duplicated; G1 is the gap between M and S; G2 is the
gap between S and M. A complex of cyclin and cyclin-dependent
kinase (CDK) controls specific events of each phase. Without cyclin,
CDK is inactive. Different cyclin/CDK complexes are shown around
the cell cycle. A, B, D, and E stand for cyclin A, cyclin B, cyclin D, and
cyclin E, respectively.
Answer : A
See Figure 15-2: The cell cycle and its control system. M is the
mitosis phase, when the nucleus and the cytoplasm divide;
S is the phase when DNA is duplicated; G1 is the gap between
Mitosis
M
G
2
G
1
A/CDK1
S
DNA replication
A/CDK2
E/CDK2
D/CDK4
D/CDK6
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Death signal
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CHAPTER 15
9. In cellular apoptosis, the release of cytochrome c acti-
Molecular and Genomic Surgery
vates the:
A. FAS receptor.
B. A membrane bound death receptor.
C. Tumor necrosis factor (TNF) receptor.
D. Caspase cascade.
Death
receptor
FIG. 15-3. A simplified view of the
apoptosis pathways. Extracellular death
receptor pathways include the activation
of Fas and tumor necrosis factor (TNF)
receptors and consequent activation
of the caspase pathway. Intracellular
death pathway indicates the release of
cytochrome c from mitochondria, which
also triggers the activation of the caspase
cascade. During apoptosis, cells undergo
DNA fragmentation and nuclear and cell
membrane breakdown and are eventually
digested by other cells.
(e.g., TNF or Fas)
Death
receptor
signaling
pathway
Activation of
caspase cascade
Normal target cell
M and S; G2 is the gap between S and M. A complex of cyclin
and cyclin-dependent kinase (CDK) controls specific events
of each phase. Without cyclin, CDK is inactive. Different
cyclin/CDK complexes are shown around the cell cycle. A, B,
D, and E stand for cyclin A, cyclin B, cyclin D, and cyclin E,
respectively. (See Schwartz 11th ed., p. 486.)
Answer: D
See Figure 15-3. (See Schwartz 11th ed., Figure 15-8, p. 487.)
Plasma
membrane
Mitochondrion
Cytochrome c
release
Apoptotic target cell
Nucleus
10. All of the following are cell-surface receptors EXCEPT:
A. Transmitter-gated ion channels.
B. Seven-transmembrane-G-protein-coupled receptors.
C. Enzyme-linked receptors.
D. Adhesive receptors.
Answer: D
There are three major classes of cell-surface receptors: transmitter-gated ion channels, seven-transmembrane G-protein–
coupled receptors (GPCRs), and enzyme-linked receptors.
The superfamily of GPCRs is one of the largest families of
proteins, representing over 800 genes of the human genome.
Members of this superfamily share a characteristic seventransmembrane configuration. The ligands for these receptors
are diverse and include hormones, chemokines, neurotransmitters, proteinases, inflammatory mediators, and even sensory signals such as odorants and photons. Most GPCRs
signal through heterotrimeric G proteins, which are guanine
nucleotide regulatory complexes. Thus the receptor serves
as the receiver, the G protein serves as the transducer, and
the enzyme serves as the effector arm. Enzyme-linked receptors possess an extracellular ligand-recognition domain and a
cytosolic domain that either has intrinsic enzymatic activity
or directly links with an enzyme. Structurally, these receptors
usually have only one transmembrane-spanning domain. Of
at least five forms of enzyme-linked receptors classified by
the nature of the enzyme activity to which they are coupled,
the growth factor receptors such as tyrosine kinase receptor
or serine/threonine kinase receptors mediate diverse cellular
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11. Dysregulation of transforming growth factor-β (TGF-β)
signaling is associated with all EXCEPT:
A. Cancer.
B. Inguinal hernias.
C. Marfan syndrome.
D. Thoracic aortic aneurysm.
events including cell growth, differentiation, metabolism, and
survival/apoptosis. Dysregulation (particularly mutations) of
these receptors is thought to underlie conditions of abnormal cellular proliferation in the context of cancer. The following sections will further review two examples of growth
factor signaling pathways and their connection with human
diseases. (See Schwartz 11th ed., p. 488.)
Answer: B
Resistance to TGF-β’s anticancer action is one hallmark of
human cancer cells. TGF-β receptors and SMADs are identified as tumor suppressors. The TGF-β signaling circuit can
be disrupted in a variety of ways and in different types of
human tumors. Some lose TGF-β responsiveness through
downregulation or mutations of their TGF-β receptors. The
cytoplasmic SMAD4 protein, which transduces signals from
ligand-activated TGF-β receptors to downstream targets, may
be eliminated through mutation of its encoding gene. The
locus encoding cell cycle inhibitor p15INK4B may be deleted.
Alternatively, the immediate downstream target of its actions,
cyclin dependent kinase 4 (CDK4), may become unresponsive
to the inhibitory actions of p15INK4B because of mutations
that block p15INK4B binding. The resulting cyclin D/CDK4
complexes constitutively inactivate tumor suppressor pRb by
hyperphosphorylation. Finally, functional pRb, the end target
of this pathway, may be lost through mutation of its gene. For
example, in pancreatic and colorectal cancers, 100% of cells
derived from these cancers carry genetic defects in the TGF-β
signaling pathway. Therefore, the antiproliferative pathway
converging onto pRb and the cell division cycle is, in one way or
another, disrupted in a majority of human cancer cells. Besides
cancer, dysregulation of TGF-β signaling also has been associated with other human diseases such as Marfan syndrome and
thoracic aortic aneurysm. (See Schwartz 11th ed., p. 489.)
CHAPTER 15
Molecular and Genomic Surgery
12. All of the following are true regarding type 2 diabetes
EXCEPT:
A. More than 90% of individuals have insulin resistance.
B. Genetic mutation in the cell-surface insulin receptors
(InsR) cause the disease.
C. Majority of cases may result from defects in down-
stream-signaling components in the insulin-signaling pathway.
D. Phosphoryl group is added by the insulin receptor
substrate (IRS).
FIG. 15-4. Insulin-signaling pathway. Insulin is a
peptide growth factor that binds to and activates
the heterotetrameric receptor complex (InsR). InsR
possesses protein tyrosine kinase activity and is able
to phosphorylate the downstream insulin receptor
substrate (IRS). Phosphorylated IRS serves as a scaffold
and controls the activation of multiple downstream
pathways for gene expression, cell survival, and glucose
metabolism. Inactivation of the insulin pathway can
lead to type 2 diabetes.
Answer: D
See Figure 15-4. Insulin is a peptide growth factor that binds
to and activates the heterotetrameric receptor complex
(InsR). InsR possesses protein tyrosine kinase activity and is
Insulin
receptor
(InsR)
Adaptor
MAPK
cascade
Nucleus
Insulin
IRS
Lipid & glucose
metabolism
Plasma
membrane
PI3K
Cell
survival
Gene
expression
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CHAPTER 15
13. Which of the following statements are TRUE?
Molecular and Genomic Surgery
A. In normal cells, oncogenes promote cell growth by
activating cell cycle progression.
B. In cancer cells, oncogenes promote cell growth by
activating cell cycle progression.
C. Tumor suppressors enhance oncogene function.
D. Oncogenes do not play an important role in main-
taining controlled state of cell growth.
14. All of the following are correct about human embryonic
stem cells (hESCs) EXCEPT:
A. They are derived from early preimplantation
embryos.
B. They are derived from blastocysts.
C. They can generate all differential germ layers.
D. They are considered multipotent.
able to phosphorylate the downstream IRS. Phosphorylated
IRS serves as a scaffold and controls the activation of multiple
downstream pathways for gene expression, cell survival, and
glucose metabolism. Inactivation of the insulin pathway can
lead to type 2 diabetes. (See Schwartz 11th ed., Figure 15-10,
p. 489.)
Answer: A
There are two classes of cancer genes in which alteration has
been identified in human and animal cancer cells: oncogenes,
with dominant gain-of-function mutations, and tumor suppressor genes, with recessive loss-of-function mutations. In
normal cells, oncogenes promote cell growth by activating
cell cycle progression, whereas tumor suppressors counteract
oncogenes’ functions. Therefore, the balance between oncogenes and tumor suppressors maintains a well-controlled
state of cell growth. (See Schwartz 11th ed., p. 490.)
Answer: D
hESCs are derived from early preimplantation embryos
called blastocysts (5 days postfertilization) and are capable of
generating all differentiated germ layers in the body by chimera assays or 2-D/3-D differentiation in a dish—ectoderm,
mesoderm, and endoderm—and therefore are considered
pluripotent. There are two pluripotent states associated with
hESCs, one of which is the classic culture with basic fibroblast
growth factor (bFGF) and knock out replacer (KSR), termed
as “primed” pluripotent state. More recently, “naive” hESC
culture methods have been introduced based on mouse studies, by supplementing 2i inhibitors (MEK1 and GSK3β inhibitors) into the medium in addition to bFGF. (See Schwartz
11th ed., p. 492.)
15. Gene expression detection method that provides information regarding mRNA size is:
A. Polymerase chain reaction.
B. Southern blot hybridization.
C. Northern blot hybridization.
D. Immunoblotting.
Answer: C
Northern blotting refers to the technique of size fractionation
of RNA in a gel and the transferring of an RNA sample to a
solid support (membrane) in such a manner that the relative
positions of the RNA molecules are maintained. The resulting
membrane then is hybridized with a labeled probe complementary to the mRNA of interest. Signals generated from
detection of the membrane can be used to determine the size
and abundance of the target RNA. In principle, Northern blot
hybridization is similar to Southern blot hybridization (and
hence its name), with the exception that RNA, not DNA, is
on the membrane. Although reverse-transcriptase PCR has
been used in many applications, Northern analysis is the only
method that provides information regarding mRNA size and
has remained a standard method for detection and quantitation of mRNA. The process of Northern hybridization
involves several steps, as does Southern hybridization, including electrophoresis of RNA samples in an agarose-formaldehyde gel, transfer to a membrane support, and hybridization
to a radioactively labeled DNA probe. Data from hybridization allow quantification of steady-state mRNA levels and, at
the same time, provide information related to the presence,
size, and integrity of discrete mRNA species. Thus, Northern
blot analysis, also termed RNA gel blot analysis, commonly is
used in molecular biology studies relating to gene expression.
(See Schwartz 11th ed., p. 494.)
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16. Which of the following drugs is an example of an immunotherapy that targets an oncogene?
A. Trastuzumab
B. Methotrexate
C. Adriamycin
D. Gleevec
17. All of the following are TRUE regarding Bifunctional
RNAi Technology EXCEPT:
A. Field has worked to define oncogene and nononco-
gene addiction.
B. It advances the understanding of discrimination
between driver and passenger genes.
C. The field made strides in appreciation of complexity
of network interaction.
D. The knowledge in the field has been effectively and
reproducibly clinically translated.
Answer: A.
One of the most exciting applications of immunotherapy has
come from the identification of certain tumor targets called
antigens and the aiming of an antibody at these targets. This
was first used as a means of localizing tumors in the body
for diagnosis and was more recently used to attack cancer
cells. Trastuzumab (Herceptin) is an example of such a drug.
Trastuzumab is a monoclonal antibody that neutralizes the
mitogenic activity of cell-surface growth factor receptor HER2, which is overexpressed in approximately 25% of breast
cancers. HER-2–overexpressing tumors tend to grow faster
and generally are more likely to recur than tumors that do
not overproduce HER-2. Trastuzumab is designed to attack
cancer cells that overexpress HER-2 by slowing or preventing the growth of these cells, resulting in increased survival
of HER-2–positive breast cancer patients. (See Schwartz 11th
ed., p. 491.)
Answer: D
Over the last 20 years, the field has worked to define oncogene and nononcogene addiction, discriminate between
driver and passenger genes, and appreciate the complexity of
complex, robust, network interactions. These insights have
led to a preliminary understanding of therapeutically relevant
sensitivity and resistance pathway signal patterns requiring
multiple target modulation. However, this knowledge has not
been effectively or reproducibly clinically translated. Clinical
response is usually far greater when a combination of singletarget molecular therapy is administered. However, it must
also be realized that targeting two or more pathways may also
increase the toxicity profile, particularly if target specificity is
limited. When attempted, off-target toxicity has been demonstrated with combination small-molecule therapy. In contrast, multitargeting bifunctional short hairpin (bi-shRNA)
DNA vectors are designed to limit off-target effect given the
high specificity for the genes they are designed to target. (See
Schwartz 11th ed., p. 505.)
CHAPTER 15
Molecular and Genomic Surgery
18. Which of the following about CRISPR is TRUE?
A. CRISPR-Cas9 technology can be used to edit single
genes through gene knockout, mutation, and addition of an epitope tag.
B. A typical CRISPR region contains a cluster of DNA
repeats interspersed with spacers.
C. CRISPR-Cas9 mediated gene editing involves DNA
break and repair.
D. All of the above.
Answer: D
CRISPR stands for clustered regularly interspaced short palindromic repeats. It is a region on the genomic DNA first
discovered in the microbes as an adapted immune system
against exogenous DNA. A typical CRISPR region contains
a cluster of short (21–48 bp) DNA repeats (ranging from
2 to 100) interspaced by nonrepetitive sequences called
spacers. Within a CRISPR region, while each spacer has its
unique sequence, the sequence of the repeats is highly conserved. Several genes, called the CRISPR-associated (Cas)
genes, are almost always found directly flanking the CRISPR
region. Currently, CRISPR-Cas9 is most used for editing
single genes, through gene knockout, gene mutation, or the
addition of an epitope tag to a native gene, for functional characterization of the gene of interest. For example, oncogenes or
tumor suppressor genes can be knocked out to identify the
causative gene for a particular cancer type; point mutations
in functional domains may illustrate the mechanism of action
of a protein; for proteins without available antibodies, epitope
tags can be inserted onto the native gene for the detection of
the native protein. (See Schwartz 11th ed., p. 506.)
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PART II
Specific Consideration
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