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VOL 11 SUPPLEMENT 1 PP S1–XXX

November 2004 Volume 11 Supplement 1

Teaching
ACADEMIC RADIOLOGY

Research

Patient Care
A Primer on Molecular
OFFICIAL
JOURNAL OF Biology for Imagers
Association of University
Radiologists
Society of Chairmen of
Academic Radiology
Departments

Association of Program
Directors in Radiology

American Association of
Academic Chief Residents Editor:
in Radiology King C.P. Li, MD, FRCP(C), MBA

Alliance of Medical Student

Educators in Radiology Publication of this supplement is made possible by
educational grants from Berlex, Inc. and Siemens
NOVEMBER 2004

Radiology Research Alliance Medical Solutions.

Radiology Alliance for Health

Services Research

www.academicradiology.org
 November 2004
Volume 11 Supplement 1
䊚 AUR, 2004

The Official Journal of the Association of University Radiologists, the Society of Chairmen of Academic Radiology Departments,
the Association of Program Directors in Radiology, the American Alliance of Academic Chief Residents in Radiology, Alliance of
Medical Student Educators in Radiology, Radiology Research Alliance, and Radiology Alliance for Health Services Research

EDITOR-IN-CHIEF ASSOCIATE EDITORS

Stanley Baum, MD Academic Radiology Editorial Office Tsutomu Araki, MD Tokyo, Japan
University of Pennsylvania Mostafa Atri, MD Toronto, Ontario
3600 Science Center, Suite 370 Dennis M. Balfe, MD St Louis, MO
3600 Market Street
Johan G. Blickman, MD, PhD Nijmegen, the Netherlands
Philadelphia, PA 19104
Alan S. Brody, MD Cincinnati, OH
Mauricio Castillo, MD Chapel Hill, NC
Claudia da Costa Leite, MD Sao Paulo, Brazil
EXECUTIVE ASSOCIATE EDITOR Adrian K. Dixon, FRCR, MbCHb Cambridge, England
Felix Sze-Kway Chew, MD Winston-Salem, NC Laurie L. Fajardo, MD Iowa City, IA
Howard P. Forman, MD, MBA New Haven, CT
TERM Philippe A. Grenier, MD Paris, France
DEPUTY EDITORS ENDS Man Chung Han, MD Seoul, Korea
Victor M. Haughton, MD Madison, WI
C. Craig Blackmore, MD, MPH Seattle, WA 2005 Lawrence E. Holder, MD Jacksonville, FL
Jannette Collins, MD, MEd Madison, WI 2004 Ferenc A. Jolesz, MD Boston, MA
N. Reed Dunnick, MD Ann Arbor, MI 2004
Constantine Gatsonis, PhD Providence, RI 2006 Harold L. Kundel, MD Philadelphia, PA
William R. Hendee, PhD Milwaukee, WI 2006 Curtis P. Langlotz, MD, PhD Philadelphia, PA
Eric A. Hoffman, MD Iowa City, IA 2005 Guy Marchal, MD Leuven, Belgium
Ella A. Kazerooni, MD Ann Arbor, MI 2004 Gerhard H. Mostbeck, MD Vienna, Austria
Heinz U. Lemke, PhD Berlin, Germany 2006 Suresh K. Mukherji, MD Ann Arbor, MI
Robert Lenkinski, PhD Boston, MA 2006
C. Leon Partain, MD, PhD Nashville, TN
King C. P. Li, MD Bethesda, MD 2005
Robert F. Mattrey, MD San Diego, CA 2006 Paolo Pavone, MD Rome, Italy
Reuben S. Mezrich, MD, PhD Baltimore, MD 2004 Hans Ringertz, MD, PhD Stockholm, Sweden
Etta Pisano, MD Chapel Hill, NC 2005 Arvin E. Robinson, MD Rochester, NY
Scott O. Trerotola, MD Philadelphia, PA 2006 Michael R. Sage, MD South Australia, Australia
Ralph Weissleder, MD, PhD Boston, MA 2006 Steven E. Seltzer, MD Boston, MA
Beverly P. Wood, MD, MSc Los Angeles, CA 2005
Wilbur L. Smith, MD Detroit, MI
Brent K. Stewart, PhD Seattle, WA
Henrik S. Thomsen, MD Herlev, Denmark
EDITORS EMERITI Thomas Vogl, MD Frankfurt, Germany
Edmund A. Franken, Jr, MD Iowa City, IA Gerald L. Wolf, PhD, MD Boston, MA
Bruce J. Hillman, MD Charlottesville, VA

EDITORIAL EXECUTIVE COMMITTEE
Ronald L. Arenson, MD
Felix Sze-Kway Chew, MD
N. Reed Dunnick, MD
Laurie L. Fajardo, MD
G. Scott Gazelle, MD, MPH, PhD
Ferenc A. Jolesz, MD
Etta D. Pisano, MD
Steven E. Seltzer, MD
H. Dirk Sostman, MD
EDITORIAL COORDINATOR
Flora F. Cauley
EDITORIAL CONSULTANTS
Kevin Berbaum, PhD
2004 Michael A. Bettmann, MD
2008
Robert C. Brasch, MD
2005
2005 R. Edward Coleman, MD
2007 Emily Conant, MD
2006 Elliot K. Fishman, MD
2006 Maryellen L. Giger, PhD
2008 David Gur, ScD
2005
Claudia I. Henschke, MD, PhD
Charles B. Higgins, MD
Kenyon K. Kopecky, MD
Elizabeth Krupinski, PhD
Philadelphia, PA Elvira V. Lang, MD
Scott A. Mirowitz, MD
Michael J. Pentecost, MD
Deborah J. Rubens, MD
Melvyn H. Schreiber, MD
Joanne J. Seibert, MD
Lorraine G. Shapeero, MD
Robert L. Siegle, MD
H. Dirk Sostman, MD
Stephen J. Swensen, MD
Lee B. Talner, MD
William M. Thompson, MD
Stuart W. Young, MD
 Volume 11
Supplement 1
November 2004

Editor’s Note Chapter V

King C.P. Li S1 A Primer on Molecular Biology for S54
Imagers: V. Genomics: A Global Approach
Foreword to the Study of Genes
Kien Vuu, Sunil D. Pandit, King C.P. Li
Belinda Seto S2

Chapter VI
Preface
A Primer on Molecular Biology for S66
Molecular Imaging and Molecular Biology S5 Imagers: VI. Proteomics: The Large-Scale
James H. Thrall Study of Proteins
Joseph T. Azok, Sunil D. Pandit, King C.P. Li
Chapter I
A Primer on Molecular Biology for S7 Chapter VII
Imagers: I. DNA, How Does It Work? A Primer on Molecular Biology for S77
Sunil D. Pandit, Mark Bednarski, King C.P. Li Imagers: VII. Molecular Imaging Probes
S. Narasimhan Danthi, Sunil D. Pandit,
Chapter II King C.P. Li

A Primer on Molecular Biology for S16

Imagers: II. Transcription and Gene Chapter VIII
Expression A Primer on Molecular Biology for S85
Sunil D. Pandit, King C.P. Li Imagers: VIII. Equipment for Imaging
Molecular Processes
Chapter III David M. Thomasson, Ahmed Gharib,
King C.P. Li
A Primer on Molecular Biology for S28
Imagers: III. Proteins: Structure and
Chapter IX
Function
Sunil D. Pandit, King C.P. Li A Primer on Molecular Biology for S97
Imagers: IX. How to Become a “Molecular
Imager”
Chapter IV
King C.P. Li
A Primer on Molecular Biology for S42
Imagers: IV. Concepts and Basic Methods Subject Index S101
in Molecular Biology
Sunil D. Pandit, King C.P. Li Author Index S108

(Continued)
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Radiologists, the Society of Chairmen of Academic Radiology Departments,
the Association of Program Directors in Radiology, and the American Association
of Academic Chief Residents in Radiology. The journal invites well-formulated
original reports of clinical and laboratory investigations in diagnostic imaging, the
diagnostic use of radioactive isotopes, computed tomography, positron emission
tomography, magnetic resonance imaging, ultrasound, digital subtraction angiog-
raphy, and related techniques. A special section is devoted to publication of very
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published.

This series of articles was developed as a result of my
discussion with many leaders in the fields of molecular
imaging and academic radiology. There was a perceived
need for educational materials that could help imaging
scientists and radiologists acquire knowledge of molecular
biology. Although there is an abundance of educational
material on molecular biology, none was written with the
imaging scientists as the target audience. This is an at-
tempt to fulfill this need. This series is designed to give
readers with little to no background in molecular biology
sufficient information so that they can follow current liter-
ature and share the excitement of the rapid advances en-
abled by the sequencing of the human genome and other
technological breakthroughs. Many excellent references
and web sites are included in the bibliography so that
interested readers can delve deeper into topics that inter-
est them. Glossaries are also included in many of the arti-
cles to facilitate the learning of the jargon, which is a
critical first step in learning about a new scientific field.
The articles in this series should be read in the sequence
that they appear since the later articles build on knowl-
edge gained from earlier articles. The many contributors
of this series were all members of the Imaging Sciences
Program in the Clinical Center of the National Institutes
Acad Radiol 2004; 11S:1
1 Department Radiology and Imaging Sciences, National Institutes of
Health, Bethesda, MD
© AUR, 2004
doi:10.1016/j.acra.2004.10.054
Editor’s Note
King C.P. Li, MD, FRCP(C), MBA1
of Health. Without their dedication, expertise, and hard
work this series would not have come to fruition. Special
thanks should go to Dr. Sunil Pandit, who shouldered a
lot of the burden in the preparation of the articles and in
assuring the accuracy of the information. A lot of credit
should also go to Dr. Stanley Baum, the Editor-in-Chief
of Academic Radiology, the Editorial Board of Academic
Radiology, and the Board of the Association of Univer-
sity Radiologists for supporting this project. Since there
are a limited number of pages in each issue of Academic
Radiology, each page must be wisely used. We are grate-
ful that a substantial number of journal pages have been
allocated to this project. We hope that the readers would
agree that we have used this precious resource wisely.
The idea of turning this series of articles into a mono-
graph originated from Dr. Stanley Baum. In order to
reach a broader audience it was decided that the mono-
graph should be distributed for free. Dr. Baum was instru-
mental in finding the sponsors for funding this project.
We would like to take this opportunity to thank Siemens
Medical Systems, Inc. and Berlex Laboratories for spon-
soring this project. Without their generous support we
would not be able to produce and distribute this mono-
graph. The preparation of this series is a learning process
for all of us, so please feel free to contact me regarding
potential improvements that we can make in future edi-
tions of this monograph. Finally, I wish you the best in
your journey into the wonderful world of molecular biol-
ogy and molecular imaging.
S1

Research into the pathophysiology of human diseases is
performed across multiple biomedical disciplines and re-
lies on observations recorded in both the clinic and the
laboratory. In the clinic, radiologists are continually
searching for new and improved methods to image pa-
tients and capture the presence and stage of disease.
Methods currently available use images at the anatomical
level of the organ or tissue; however, efforts to visualize
disease and biological processes at a more fundamental
level, eg, molecular imaging, are gaining momentum, as
heralded by technologies that investigate the expression of
genes and proteins in vivo.
Understanding the aberrant expression of genes and
proteins has long been a fundamental aspect of biomedi-
cal research. In some cases, the relationship between a
mutated gene and a particular disease process can be in-
ferred on the basis of genetic and biochemical studies
alone. One such example is cystic fibrosis, where a muta-
tion in the gene encoding for a protein key to the trans-
membrane transportation of salt results in catastrophic
lung disease. Insight into the basis for this disease was
driven primarily by in vitro experiments, first examining
the genetic makeup of cystic fibrosis patients and then
testing the biochemical function of the protein involved in
disease. The discovery of dozens of similar relationships
between gene mutation and disease has fueled the phar-
maceutical industry’s efforts to understand the molecular
biology of disease and manipulate it for the patient’s ben-
efit.
However, molecular analysis alone may not be suffi-
cient for the understanding of more complex diseases,
like cancers, where multiple mutations to tumor suppres-
sor genes or oncogenes hinder the repair of damaged
DNA and encourage uncontrolled cell growth. In such
settings, it becomes valuable to understand the dynamic
interplay between disease and biology in the living pa-
tient. The advent of molecular imaging allows researchers
to manipulate principles of molecular biology and apply
them to radiology in order to enhance the ability to image
disease and understand pathophysiology. With molecular
imaging, the static snapshots of yesterday’s in vitro stud-
ies of gene expression now yield to a highly dynamic
S2
Foreword
Belinda Seto, PhD
view of how defunct genes and proteins change biochemi-
cal/molecular pathways to create disease in vivo. More-
over, molecular imaging methods have the potential to
bring a new level of resolution to the staging of disease
and the tracking of its progression.
As this series of articles demonstrates, the field of mo-
lecular imaging has its basis in nuclear medicine in that it
relies on injection of contrast materials to image an in
vivo process. However, unlike previous technologies, the
contrast agents used in molecular imaging specifically
bind to genes or proteins of interest (by taking advantage
of the specificity of cell surface receptors or proteins),
thereby addressing questions of function at the subcellular
level. The presence or activity of a gene or protein can be
reported by a variety of probes in the form of fluores-
cence, luminescence, radioactivity, or the activity of para-
magnetic atoms, as discussed in Chapter VII, “Molecular
Imaging Probes.” The resulting signal can then be imaged
through a variety of modalities (eg, CT, MRI, SPECT, or
PET), as outlined in Chapter VIII of the series “Equip-
ment for Imaging Molecular Processes.” For example,
researchers can study a particular gene by linking it to
genes for proteins with luminescent properties (such as
luciferase). Expression of the target gene is thereby cou-
pled to the release of the reporter’s luminescence, and the
presence or function of the protein of interest is illumi-
nated. The primary challenge presented by this system is
to limit the amount of nonspecific background signal (ie,
ensuring that luminescence only occurs when the target
has been attained). However, as discussed in Part VIII,
this problem may be reduced by the use of “smart
probes,” single-stranded DNA strands connected to bea-
con fluorochromes, quenchers, and proteases. The quench-
ers utilized in this system play a key role in limiting non-
specific signal by blocking fluorochrome emission until
the probe has bound to its target. Once the target has
been attained, proteases degrade the quencher, allowing
the fluorochrome to emit light. Similar concepts are being
used with other types of probes and in various imaging
modalities to overcome the challenge of nonspecific bind-
ing and image a larger array of processes.
 Academic Radiology, Vol 11, Suppl 1, November 2004
Because molecular imaging is a relatively new field,
significant limitations in technology must still be over-
come. These include the need for contrast agents or
probes that are biocompatible and the need for an ade-
quate delivery method that allows imaging materials to
interact with proteins/genes of interest long enough for
their presence to be detected. Moreover, imaging intracel-
lular processes remains a challenge since it is unclear
how to construct imaging materials that enter living cells
without destroying them. An additional challenge is pre-
sented by the need to expand the signal from a gene of
interest (which is often present in very small amounts) to
detect it. Thus, the key issue in the field remains how to
design imaging modalities to detect signals that are inher-
ently transient or weak. The application of this field to
living human subjects will require a combined expertise
in molecular biology and radiology; therefore, the purpose
of this series is not only to introduce the field, but also to
provide radiologists with a primer on the fundamentals of
molecular biology.
The initial articles in this series guide readers through
a simple refresher of the principles of molecular biology.
At its core, molecular biology studies the basic structure
of DNA, the transcription of DNA into RNA, and the
final translation of RNA into protein. In part I of the se-
ries (“DNA, How Does It Work?”), readers learn the
components of DNA, the structure of its double helix, and
the manner in which it exists in chromosomes. Readers
also learn the difference between eukaryotic and prokary-
otic genomes and the basis of the genetic code. Finally,
the article concludes with a brief discussion of DNA rep-
lication, including the mechanics and enzymes involved
and the ingenious method of semiconservative replication
that minimizes replication errors.
In the next article (“Transcription and Gene Expres-
sion”), readers learn about the first step in the production
of proteins, namely, the transcription of DNA into RNA.
The complex mechanism by which certain genes are pre-
pared for transcription into RNA (using gene promoters)
is broached in this article, and the methods of transcrip-
tion are compared for prokaryotes and eukaryotes. Once
this process of transcription is complete, RNA (the inter-
mediary, or messenger, of protein production) transports
the genetic code for a particular protein from the nucleus
to the cytoplasm of the cell for refinement and translation
into protein. These critical steps to protein production,
which are important in the design of molecular imaging
probes, are discussed at length in this article.
FOREWORD
The article entitled “Proteins: Structure and Function”
introduces amino acids (the building blocks of proteins)
and demonstrates how the structure and function of a pro-
tein are intimately entwined. The article also describes the
differences between the prokaryotic and eukaryotic ribo-
somes responsible for protein production and the roles of
ribosomal RNA (rRNA) and transfer RNA (tRNA). Read-
ers are guided through the process by which the messen-
ger RNA signal is converted to a protein (translation) and
the final processing of the protein into its functional form.
In this article, methods particularly important to the pro-
duction of imaging proteins are described. Specifically,
methods to quantify amounts of proteins, determine their
structure and sequence, and construct them in vitro are
introduced. Finally, the authors apply basic information
about the structure of proteins to describe antibodies, a
particular type of protein with exquisite specificity for
other molecules and great potential as a probe for imag-
ing experiments.
Although the theory behind molecular biology con-
cepts will be useful to many radiologists as they design
experiments, an understanding of the commonly used
methodology will be required for production of probes.
These matters are discussed in this series as well (see
“Concepts and Basic Methods in Molecular Biology”).
First, this article introduces basic techniques to isolate
DNA, RNA, and proteins from cells and tissues. Next, the
use of restriction endonucleases to cut DNA is covered,
and the isolation of resulting DNA fragments by gel elec-
trophoresis is introduced. This crude imaging and purifi-
cation technique takes advantage of the size and charge of
varying DNA fragments to separate them on a charged
gel matrix. The separated fragments of DNA may then be
incorporated into expression vectors so that researchers
can amplify gene products of interest in bacteria or eu-
karyotic cells. In this manner, many of the reporter con-
structs used in molecular imaging can be created. In addi-
tion to the aforementioned techniques, the article intro-
duces southern, northern, and western blotting, which are
used to detect and isolate DNA, RNA, and protein (re-
spectively) for use in recombinant DNA libraries.
With a better appreciation of the mantra of molecular
biology (DNA to RNA to protein) and the technologies to
manipulate molecular processes, the novice molecular
imager may now explore the totality of the genetic code
and the body of proteins it encodes. The article entitled
“Genomics: A Global Approach to the Study of Diseases”
introduces genomics (the study of genomes) and its impli-
cations for understanding the human genome and how the
S3
 BELINDA SETO
expression of genes changes with disease. With the map-
ping of the human genome complete, researchers now
have the basis to determine the function of the genes
identified. Efforts to define protein function may soon
rely on molecular imagers, since they have many of the
tools necessary to directly visualize protein dynamics in
vivo. This article explores the implications to health and
society of this amazing leap.
Because molecular imaging is a relatively new field,
current studies still rely on small animal models rather
than humans. Nevertheless, examples from small animal
systems provide a glimpse into the types of studies that
may be performed in humans in the near future. For ex-
ample, using a luciferase reporter, the activity of tumor
suppressor proteins (like p53) can be investigated in mice
before and after exposure to tumorigenic agents. Using
this system, molecular imagers have found that p53 is
increased constitutively in tumor cells in vivo. This exam-
ple illustrates how molecular imaging can elucidate the
pathophysiology behind the development of cancer, and
S4
Academic Radiology, Vol 11, Suppl 1, November 2004
provides a model to study carcinogenesis in humans. An-
other example of the application of molecular imaging
assesses the efficacy of doxorubicin in the treatment of
lymphoma in a mouse model. In this model, mice are
injected with a lymphoma cell line expressing fluorescent
molecules. After an incubation period, radiolabeled an-
nexin V is injected as a specific probe for dying cells. By
imaging the cells before and after exposure to doxorubi-
cin (using SPECT), the percentage of cell death affected
by the chemotherapeutic drug can be ascertained.
The implications of these modalities are obvious. Mo-
lecular imaging not only enhances our understanding of
disease pathophysiology, but it can also guide the devel-
opment of therapies and assessment of treatment efficacy.
In the process, researchers can gain a finer understanding
of genetic and biological processes. In the world of
“Dolly” the genetically cloned sheep, molecular biology
is already appreciated as a tool of the future. Now the
potential and power of combining molecular biology with
imaging is opening “eyes” even further.

Molecular Imaging and Molecular Biology1
Developments in molecular biology and molecular genet-
ics from the past three decades have provided medical
science with the increasing ability to understand the mo-
lecular basis of life. In radiology these breakthroughs
have led in part to the concept of molecular imaging, and
it is through molecular imaging that radiology will play a
major role in the age of molecular medicine.
To beneficially incorporate molecular imaging methods
into practice, radiologists will need to acquire additional
knowledge about molecular biology (1– 4). The principles
of molecular biology encompass the building blocks for
development of novel molecular imaging pharmaceuticals
and for understanding molecular imaging applications.
In this Supplement of Academic Radiology, Dr. King
Li and his colleagues provide a series of review articles
on molecular biology for imagers. Taken together the se-
ries constitutes a primer on molecular biology tailored for
the needs of radiologists. This is a major and important
undertaking on behalf of the specialty. Drs. Li and his
colleagues deserve both our congratulations and our
thanks for this outstanding contribution. These articles
should provide radiologists with the basic science frame-
work for approaching molecular imaging. Dr. Li and his
colleagues logically start with a discussion of the princi-
ples of DNA replication and function.
Predicting the future is a hazardous undertaking at
best, but it is not too soon to predict that molecular imag-
ing methods will significantly expand the horizons of
medical imaging and will have remarkable positive influ-
ences on the ability to diagnose and treat disease. Simply
Reprinted with permission from Acad Radiol 2004; 11:S5–S6
1 From the Department of Radiology, Massachusetts General Hospital, Har-
vard Medical School, MZ-FND 216, 14 Fruit Street, Boston, MA. Address
correspondence to J.H.T.
© AUR, 2004
doi:10.1016/j.acra.2004.10.011
Preface
James H. Thrall, MD
stated, molecular derangements occur at the very begin-
ning of disease processes whereas anatomically detectable
abnormalities that have been the historic basis of radiol-
ogy practice present themselves much later. Molecular
imaging methods will permit earlier diagnosis and offer
the further promise of improved diagnostic specificity.
That is, the underexpression or overexpression of certain
molecules is found in almost every disease, and these
changes can be used to diagnose and characterize disease
through molecular imaging.
Although the term “molecular imaging” is relatively
new, the underlying concept has been the basis of many
nuclear medicine procedures for over half a century. The
first clinically important nuclear medicine techniques were
the I-131 thyroid uptake test and the I-131 thyroid scan.
These procedures are exemplars for molecular imaging.
That is, the in vivo distribution of the molecule radioio-
dine is localized both spatially (scan) and temporally (up-
take). Fluorodeoxyglucose (FDG) PET imaging is another
important example from nuclear medicine that is finding
increasing clinical application.
Nuclear medicine also provides the generic paradigm
for the development of molecular imaging agents or
probes. That is, every radiopharmaceutical has one com-
ponent or moiety that confers tissue localization and a
second component or moiety that confers detectability.
Each molecular imaging pharmaceutical, whether de-
signed for use with magnetic resonance imaging, optical
imaging, or ultrasound, also incorporates these two differ-
ent functional components to permit both specific tissue
localization and efficient detection by the respective imag-
ing modality. From this standpoint, molecular imaging is
actually an old friend to radiologists with many of the
principles of radiotracer uptake and localization being
applied to MRI, optical, and ultrasound applications.
But the power of molecular imaging will now increas-
ingly come from the ability to harvest the learning of molec-
S5
 THRALL
ular biology. Theoretically, it is now possible to design a
molecular imaging agent to target each step in the sequence
from DNA replication to protein synthesis and subsequently
all the steps of protein metabolism. This is indeed a power-
ful concept that holds the promise of disease phenotyping
and early assessment of therapeutic efficacy.
From this it is a logical step to the vision of “personal-
ized medicine,” where disease phenotyping will be used
to tailor the most optimum therapeutic regimen patient by
patient. A well-known current example of this outside of
imaging is the use of Her-2/neu gene expression as an
indicator of whether breast cancer patients will respond to
the drug Herceptin, a monoclonal antibody. Only patients
with the right phenotype respond to the drug.
Molecular imaging promises to become a powerful
new addition to the armamentarium of medical imaging.
S6
Academic Radiology, Vol 11, Suppl 1, November 2004
Radiologists will be well served to acquire the knowledge
necessary to incorporate these new methods into their
practices, including knowledge of fundamental principles
in molecular biology. This Supplement offer the opportu-
nity to do this in a very efficient way that emphasizes the
links between basic science principles and the potential
for future clinical applications.
REFERENCES
1. Meade T. Seeing is believing. Acad Radiol 2001;8:1–3.
2. Luker GD, Piwnica-Worms D. Beyond the Genome: Molecular imaging
in vivo with PET and SPECT. Acad Radiol 2001;8:4 –14.
3. Bremer C, Weissleder R. In vivo imaging of gene expression: MR and
optical technologies. Acad Radiol 2001;8:15–23.
4. Wagenaar DJ, Weissleder R, Hengerer A. Glossary of molecular imag-
ing terminology. Acad Radiol 2001;8:409 – 420.

A Primer on Molecular Biology for Imagers:
Sunil D. Pandit, PhD, Mark Bednarski, MD, PhD, King C.P. Li, MD, MBA
In the past 20 years, tremendous progress has been made
in the field of molecular biology. Since the discovery of
DNA structure half a century ago, we have made huge
leaps. The complete sequence of the human genome has
been determined and there are about 40,000 genes in the
human genome. Several research groups and pharmaceuti-
cal companies all over the world are targeting the various
genes for treatment of numerous diseases that affect man-
kind and are trying to elucidate the molecular mecha-
nisms that operate inside a cell. Additionally, research
groups are trying to identify unique genes, or proteins
that are potentially differentially expressed in various dis-
eases, the early detection of which can most certainly
improve prognosis for the patient. Furthermore, several
new disciplines have sprung up, which have their own
definitions and specific areas that they are studying. The
“omics” revolution began with genomics about 15 years
ago and terms such as functional genomics, proteomics,
pharmacogenomics, metabolomics, and phenomics have
been coined that relate to studying the genome, proteome,
pharmacologic variation in drug response/drug metabo-
lism, analysis of metabolites, and variation in phenotypes.
The Roslin Institute in Edinburgh reported the birth of
Dolly the lamb, the first mammal to be cloned from an
adult using the modern techniques of transgenic cloning.
The successful cloning of Dolly suggested the possibility
that similar techniques could be used to clone humans. In
1999, the first human chromosome was sequenced. Re-
searchers in the Human Genome Project reported the
Reprinted with permission from Acad Radiol 2003; 10:1215–1223
1 From the Molecular Imaging Laboratory, Imaging Sciences Program, Clini-
cal Center, 10/1N306, National Institute of Health, 9000 Rockville Pike, Be-
thesda, MD 20892 (S.D.P., M.B., K.C.P.L.). Received July 15, 2003; ac-
cepted July 15. Address correspondence to S.D.P.
© AUR, 2003
doi:10.1016/j.acra.2004.10.002
Chapter I
I. DNA, How Does It Work?1
complete sequencing of the DNA making up human chro-
mosome 22. In 2000, Human Genome Project leaders
announced the completion of a “working draft” DNA se-
quence of the entire human genome. The post-genomic
era began. April 2003 marked not only the 50th anniver-
sary of the discovery of the DNA double helix but also
the completion of the human genome sequence (1,2). The
past century has witnessed a breathtaking array of discov-
eries in the biological sciences, in particular in the gen-
eral area of molecular biology, the scientific discipline
that seeks to fully understand the molecular basis of he-
redity, genetic variation, and the expression patterns of
individual units of heredity called genes.
It has become increasingly clear that the next genera-
tion of scientists and clinicians need to be trained in mul-
tiple disciplines so that they have a better understanding
of the rapid progress being made in biology. Molecular
imaging is one such area that has emerged recently,
where expertise in molecular biology, radiology, nuclear
medicine, physics, chemistry, biomedical engineering, and
pathology is required to obtain answers to many challeng-
ing questions. Never before in history were we better po-
sitioned to ask difficult questions and have the required
tools and techniques to obtain answers to them.
Even though there are many excellent books and re-
views that extensively cover the above disciplines, there
is truly a paucity of literature that radiologists can easily
refer to and educate themselves. Keeping this in mind, we
decided to write this series of reviews in an attempt to
provide a reference material for one of the disciplines
mentioned, “molecular biology,” so that radiologists and
potential “molecular imagers” could benefit from it. These
reviews are specifically geared toward educating a non-
molecular biologist about the basics of molecular biology
and how the various principles have been specifically ap-
plied to the imaging sciences. The first few articles in this
series will emphasize the basic principles of molecular
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Figure 1. Chemical structure of the four nucleotides (two with purine bases and two with pyrimidine bases) that are the funda-
mental building blocks of DNA. The sugar is called deoxyribose because it is a variation of a common sugar, ribose, which has
one more oxygen atom. (Copyright © 1999 from Griffiths AJF, Gelbart WM, Miller JH, Lewontin RC, eds. Modern genetic analy-
sis. Reproduced by permission of W.H. Freeman and Company/Worth Publishers.)

Figure 2. Three representations of the DNA double helix. (Copyright © 1999 from Griffiths AJF, Gelbart WM, Miller JH,
Lewontin RC, eds. Modern genetic analysis. Reproduced by permission of W.H. Freeman and Company/Worth
Publishers.)

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Figure 4. The genetic code. Sets of three nucleotides (codons) in

an mRNA molecule are translated into amino acids in the course of
protein synthesis according to the rules shown. The codons GUG
and GAG, for example, are translated into valine and glutamic acid,
respectively. (Copyright © 1999 from Griffiths AJF, Gelbart WM,
Miller JH, Lewontin RC, eds. Modern genetic analysis. Reproduced
by permission of W.H. Freeman and Company/Worth Publishers.)

Figure 3. Model of chromatin packing. This schematic drawing

shows some of the many orders of chromatin packing postulated
to give rise to the highly condensed mitotic chromosome. (Copy-
right © 1994 from Alberts B, Bray D, Lewis J, Raff M, Roberts K,
Watson JD, eds. Molecular biology of the cell. 3rd ed. Repro-
duced by permission of Routledge/Taylor & Francis Books Inc.)

biology and will provide a refresher for most molecular

imagers. The subsequent articles will focus on the appli-
cation of these specific principles and their use in molecu-
lar imaging.

DNA: DISCOVERY AND STRUCTURE

It was in 1865 that an Austrian Monk named Gregor
Mendel published the results of cross breeding experi-
ments he had conducted on the garden pea Pisum sati-
vum. By noting the appearance or disappearance of traits
such as pod and flower color over several generations,
Mendel was able to postulate a generalized set of rules
governing inheritance, which is now known as Mendelian
Inheritance. He proposed that there are discrete units of
Figure 5. The semiconservative replication of DNA. In each round
of replication each of the two strands of DNA is used as a template
for the formation of a complementary DNA strand. Therefore, the
original strands remain intact through many cell generations. (Copy-
right © 1994 from Alberts B, Bray D, Lewis J, Raff M, Roberts K,
Watson JD, eds. Molecular biology of the cell. 3rd ed. Reproduced
by permission of Routledge/Taylor & Francis Books Inc.)

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 PANDIT ET AL Reprinted from Academic Radiology, Vol 10, No 11, November 2003

Figure 6. A DNA replication fork and the proteins. This diagram shows a current view of how the replication proteins
are arranged at a replication fork when the fork is moving. The two-dimensional structure has been altered by folding
the DNA on the lagging strand to bring the lagging-strand DNA polymerase molecule into a complex with the leading-
strand DNA polymerase molecule. This folding process also brings the 3⬘ end of each completed Okazaki fragment
close to the start site for the next Okazaki fragment. Because the lagging-strand DNA polymerase molecule is held to
the rest of the replication proteins, it can be reused to synthesize successive Okazaki fragments; thus it is about to let
go of its completed DNA fragment and move to the RNA primer, which will be synthesized nearby, as required to start
the next DNA fragment. Note that one daughter DNA helix extends toward the bottom right and the other toward the
top left in this diagram. (Copyright © 1994 from Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD, eds. Molec-
ular biology of the cell. 3rd ed. Reproduced by permission of Routledge/Taylor & Francis Books, Inc.)

heredity (which today we call genes) that are transmitted
from generation to generation, even though some of these
units are not necessarily expressed as an observable trait
in every generation. It was in 1953, 50 years ago, that the
structure of deoxy-ribose nucleic acid (DNA) as a double
helix was first described by J.D. Watson and Francis H.C.
Crick. Since the discovery of DNA, the field of molecular
genetics has had a profound influence on our understand-
ing of molecular systems. The proposed double helix
structure of DNA was based on X-ray crystallography
studies on DNA. Francis Crick, Maurice Wilkins, and
James Watson were awarded the Nobel Prize in Medicine
and Physiology in 1962 for the pioneering work. DNA is
the primary genetic material. Genes are units of deoxyri-
bonucleic acid (DNA) contained within the chromosomes.
Nucleic acids convey genetic information. Mitosis main-
tains the parental chromosome number. Meiosis reduces
the parental chromosome number.
DNA Structure
DNA structure is that of a long, double-stranded helix.
Each strand of the helix is made of repeating nucleotide
bases of only four types: adenine (A), cytosine (C), gua-
nine (G), and thymine (T) (Fig 1). It is the sequence of
these nucleotides over the length of each individual chro-
mosome added together that make up the human genome.
The bases are from complementary classes and have dis-
tinct binding affinities for a specific complementary base.
Adenine and thymine will loosely bond to one another
when in opposite strands of the helix, as will guanine
with cytosine. The bonding between strands is accom-
plished via weak hydrogen bonds between the comple-
mentary bases, two hydrogen bonds between A and T and
three hydrogen bonds between bases G and C, which
makes the GC base pairing stronger than AT bonds. Both
DNA and RNA (ribonucleic acid) are constructed from
four main nucleotide building blocks. Each of these nu-

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Table 1
Genome Size of Model Organisms

Genome Organism Size (kb) No. of Chromosome Pairs No. of Genes

Escherichia coli Bacterium 4,700 Circular 4,000

Saccharomyces cerevisiae Yeast 13,500 Linear/16 7,000
Caenorhabditis elegans Nematode 100,000 Linear/6 25,000
Drosophila Melanogaster Fruitfly 215,000 Linear/4 27,000
Mus Musculus Mouse 3,000,000 Linear/20 36,000
Homo sapiens Human 3,000,000 Linear/23 40,000

cleotides contains a phosphate group linked to a five-car-
bon atom sugar group, which, in turn is joined to an
aromatic molecule that can either be a purine or a py-
rimidine. The building blocks of DNA and RNA are
similar except that the sugar molecule is ribose and the
base thymine is replaced by uracil in RNA. The nucle-
otides of DNA are called deoxy-ribonucleotides be-
cause they contain the sugar deoxy-ribose, where as
those of ribonucleic acid (RNA) are called ribo-nucleo-
tides because they contain the sugar ribose. A nucleo-
side contains a pentose sugar linked to either a purine
or a pyrimidine base through a C-N bond, whereas a
nucleotide is a phosphate ester of a nucleoside. Bases
A and G are called the purines and bases T, U, and C
are pyrimidines. The pyrimidine thymine is limited to
DNA, being replaced in RNA by the very similar py-
rimidine uracil. In both DNA and RNA, the nucleotides
are joined together by covalent bonds called phospho-
diester linkages, which attach phosphate group of one
nucleotide to a hydroxyl group on the sugar of the ad-
jacent nucleotide.
The Double Helix
Two separate chains of DNA are wound around each
other, each following a helical (coiling) path, resulting in
a right-handed double helix (Fig 2). The negatively
charged sugar-phosphate backbones of the molecules are
on the outside, and the planar bases of each strand stack
one above the other in the center of the helix. Between
the backbone strands run the major and minor grooves,
which also follow a helical path. The strands are joined
noncovalently by hydrogen bonding between the bases on
opposite strands to form base pairs. There are around 10
bases per turn in the DNA double helix. The two strands
are oriented in opposite directions (anti-parallel) in terms
of their 5⬘33⬘ direction, and the two strands are comple-
mentary in terms of sequence.
The Genome Of Prokaryotes and Eukaryotes
There and many differences between prokaryotes and
eukaryotes, but the key feature that distinguishes them is
the presence of a discrete, well-defined nucleus in eu-
karyotes that is absent in the prokaryotes. Additionally,
they differ in the way the genetic information is orga-
nized and processed into RNA and proteins. The prokary-
otic genome is exemplified by the Escherichia coli (E.
coli) genome, the bulk of which consists of a single cir-
cular DNA molecule of length 4.7 million bps (Table 1).
On the other hand, the length of DNA in a eukaryotic cell
depends on the species, but it is much longer than the
prokaryotic genome and is made up of number of discrete
units called chromosomes (46 in humans). All of the
DNA must be packaged into the nucleus, which is accom-
plished by the formation of a highly organized nucleopro-
tein complex called chromatin. Most of the proteins in a
eukaryotic chromatin consist of histones, of which there
are five classes: H1, H2A, H2B, H3, and H4. Histone
proteins are among the highly conserved proteins across
species, range in size from 10 –23 kd and are strongly
positively charged proteins because of the high percentage
of basic amino acids such as lysine or arginine in their
primary sequence. The nucleosome core is the basic unit
of chromosome structure and consists of a protein oc-
tamer containing two of each core histones (H2A, H2B,
H3, and H4) with 146 bps of DNA wrapped 1.8 times in
a left-handed fashion around it (Fig 3). A single molecule
of histone H1 (in some cases histone H5) stabilizes the
DNA at the point at which it enters and leaves the nu-
cleosome core and organizes the DNA between nucleo-
somes. The chromatin is organized into a larger structure
known as the 30 nm fiber that consists of left-handed he-
lix of nucleosomes with approximately six nucleosomes
per helical turn. The organization of chromatin at the
highest level shows a looped domain structure, where the
DNA in the loop is in the form of 30 nm fiber and the

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 PANDIT ET AL
loops form an array about 300 nm across. The classic
picture of paired sister chromatids at mitosis represents
the most highly condensed state of chromatin. The linear
DNA traces a single path from one tip of the chromo-
some to the other, in successive loops of up to 100 kb of
30 nm fiber anchored to the nuclear matrix in the core.
The other features that are prominent in a chromosome
are centromeres and telomeres. The centromere is the
region where the two chromatids are joined and is also
the site of attachment, via the kinetochore, to the mitotic
spindle, which pulls apart the sister chromatids at an-
aphase. Centromeres are characterized by specific short
DNA sequences although, in mammalian cells, there may
be an involvement of satellite DNA. Telomeres are spe-
cialized DNA sequences that protect the ends of linear
eukaryotic chromosomal DNA from degradation and
gradual shortening. It consists of up to hundreds of copies
of a short repeated sequence (5⬘—TTAGGG—3⬘ in hu-
mans) which is synthesized by the enzyme telomerase.
Heterochromatin comprises a portion of the chromatin in
interphase that remains highly compacted and is transcrip-
tionally inactive. The rest of chromatin (that is not hetero-
chromatin) is called euchromatin and is transcriptionally
active.
The Triplet Genetic Code
Genes are the units of inheritance from parent to child.
At a molecular level, they hold the information necessary
to direct the synthesis of cellular components. This is ac-
complished via the genetic code. The genetic information
within the DNA is conveyed by the sequence of its four
nucleotide building blocks. DNA is not the template that
directly orders the synthesis of amino acids into proteins,
rather it is the RNA called messenger RNA that carries
the message encoded by the nucleus into the cytoplasm
where it is translated into proteins. The cell contains the
means to decipher the code. Rather than read the DNA
each time a protein must be made, a copy of the gene is
made, called messenger or mRNA (ribonucleic acid), in a
process called transcription. mRNA contains a linear se-
quence of bases that are complimentary to the template
DNA strand. Base triplets (codons) in the mRNA corre-
spond to specific amino acids, which are the building
blocks of proteins (Fig 4). The triplets can be initiator or
start codons (AUG), specify one of 20 different amino
acids normally found in proteins, or act as a stop or ter-
minator codon (UAA, UAG, and UGA). The code is said
to be degenerate because more than one triplet can spec-
ify the same amino acid.
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Reprinted from Academic Radiology, Vol 10, No 11, November 2003
As the mRNA is read from one end to the other, each
triplet of bases specifies the addition of an amino acid to
the forming protein chain. The process of reading the
triplet code and formation of peptide chain is called
translation. The molecules responsible for translating the
code are made of RNA, transfer or tRNA. These mole-
cules have at one end the complimentary triplet sequence
(anti-codon) to that being read on the mRNA strand and
on the opposite end an amino acid specific to that codon.
mRNAs are usually short-lived and are destroyed in the
cytoplasm. tRNAs, however, are recycled many times.
This process of translation occurs on ribosomes, com-
posed of proteins and ribosomal RNA (rRNA), either free
in the cell cytoplasm or membrane bound. The resultant
proteins may be further processed and packaged within
the cell or be ready as is for action as enzymes, structural
elements, antibodies, or hormones. Enzymes control most
of the other chemical reactions within the cell, including
the synthesis of lipids, carbohydrates, nucleotide bases,
and other components of the cell. We will discuss the
process of transcription and translation in detail in subse-
quent review articles.
Mutations Change the Base Sequence
From the discovery that DNA is the genetic material
follows the concept that a mutation is a change in the
sequence of base pairs. Some mutations occur as a result
of normal cellular operations or interactions with the en-
vironment. These are called spontaneous mutations and
occur at a certain low frequency in any organism. Muta-
gens increase the rate of occurrence of mutations and the
changes that they cause are called induced mutations.
Any base pair of DNA can be mutated, and an alteration
that only changes a single base pair is called a point mu-
tation. A point mutation could be a transition, which
changes a purine base to purine or a pyrimidine to pyrim-
idine base (such as a G-C pair is changed to an A-T
pair). On the other hand, a transversion changes a purine
base to a pyrimidine and vice versa, so that an A-T pair
becomes a T-A or a C-G pair. Certain mutagens such as
acridines cause frame shift mutations by distorting the
structure of DNA so that additional bases are incorporated
(insertions) or omitted during replication (deletions).
Gene Structure in Prokaryotes
In prokaryotes such as bacteria, the genes are usually
grouped together in operons, which is a cluster of genes
that are related or code for enzymes in the same meta-
bolic pathway. These genes are under the control of a
 Reprinted from Academic Radiology, Vol 10, No 11, November 2003 A PRIMER ON MOLECULAR BIOLOGY FOR IMAGERS
single promoter or regulatory region. The classic example
of a well-studied operon is the lac operon described ini-
tially by Jacob and Monod. Because the structural genes
in prokaryotes are all grouped together, the transcribed
mRNA contains information for more than one protein
and is a polycistronic mRNA. The expression of most of
the genetic information in the form of polycistronic
mRNA provides the flexibility and an efficient system to
adapt rapidly to the changing environmental conditions.
Eukaryotic Genes Can Be Interrupted
The gene structure and function is much more compli-
cated in eukaryotes than in the prokaryotes. The DNA is
stored inside the nucleus and, in addition, the mitochondria
and chloroplasts (in plants) have their own separate ge-
nomes. The sequences of DNA comprising the gene in eu-
karyotes are divided into two categories, exons and introns.
The exons comprise the regions that are represented in the
messenger RNA used to produce the protein product. The
introns or intervening sequences are missing from the
mRNA and are removed by a process called splicing (to be
discussed in subsequent reviews). As discussed earlier, most
bacterial genes are organized as operons and lack the exon-
intron architecture seen in the eukaryotes.
DNA REPLICATION
The process of DNA replication is amazing. The human
genome is estimated to be about 3 billion base pairs and all
of it needs to replicated in the S phase of cell cycle every
time before a cell divides. This process is incredible and the
DNA replication machinery in the cell ensures that very few
mistakes are made in this process despite the immense size
of human DNA. An error occurs only about once in each
10 –100 billion bases. The complete process of DNA replica-
tion in human cells takes several hours, whereas the E. coli
genome is replicated in about half an hour. The single mole-
cule of DNA in E. coli genome contains 4.7 ⫻ 106 base
pairs (Table 1). DNA replication begins at a single replica-
tion origin and proceeds at about 1,000 nucleotides/second.
The process is fast and very accurate, with the error-check-
ing mechanism “proof-reading” to ensure that no error is
made in this process (error rate of 1 in 109 nucleotides in-
serted). In eukaryotes, because the genome is much larger
than prokaryotes, there are many origins of replication
(where replication can begin) that are fired up simulta-
neously, forming replication bubbles on the eukaryotic chro-
mosome.
Semi-Conservative Mode of Replication
When the replication process is complete, two DNA
molecules – identical to each other and identical to the
original – have been produced. Each strand of the original
molecule has remained intact as it served as the template
for the synthesis of a complementary strand. The replica-
tion process begins when the two complementary DNA
strands are separated. This is usually accomplished by
special proteins that unwind the molecule and expose the
nucleotide bases. This mode of replication is described as
semi-conservative because one half of each new molecule
of DNA is old and the other half is new. Because each
new strand is complementary to its old template strand,
two identical new copies of the DNA double helix are
produced during replication. This process is shown sche-
matically in Figure 5. In each new helix, one strand is the
old template and the other is newly synthesized. Because
half the original DNA molecule is saved, or conserved in
the daughter molecules, the process is called semi-conser-
vative. As a result, two identical copies now exist.
The Replication Process
As discussed previously, DNA exists in the nucleus as
a condensed, compact structure. To prepare DNA for rep-
lication, a series of proteins aid in the unwinding and sep-
aration of the double-stranded DNA molecule. These pro-
teins are required because DNA must be single-stranded
before replication can proceed. New complimentary DNA
strands are then synthesized by joining together four de-
oxy-ribonucleotide triphosphates (dATP, dGTP, dCTP,
and dTTP), one at a time, and with the removal of a di-
phosphate. Each nucleotide is selected based on its com-
plementary base pairing on the parental template strand.
The steps in DNA replication are as follows: A portion of
the double helix is unwound by an enzyme called DNA
helicase. The new DNA strand is synthesized in the 5⬘ to
3⬘ direction by DNA polymerase that catalyze the forma-
tion of a phosphor-diester bond between the 5⬘ phosphate
on an incoming nucleotide and the free 3⬘ OH on the
growing polynucleotide and additionally the hydrogen
bonds between each arriving nucleotide and the nucleo-
tides on the template strand. The new DNA strands grow
only in the 5⬘ to 3⬘ direction, and strand growth begins at
the 3⬘ end of the template and grows continuously as the
point of replication (the replication fork) moves along the
template DNA (Fig 6) (3). This is true for the parental
strand that was in 3⬘3 5⬘ orientation and the new com-
plementary strand that is being synthesized is in 5⬘33⬘
orientation and is called the leading strand. A molecule
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 PANDIT ET AL Reprinted from Academic Radiology, Vol 10, No 11, November 2003
of a DNA polymerase binds to one strand of the DNA
and begins moving along it in the 3⬘ to 5⬘ direction, using
it as a template for assembling a leading strand of nucleo-
tides and reforming a double helix. In eukaryotes, this
molecule is called DNA polymerase ␦ (delta). The DNA
polymerase then adds nucleotides one by one in an ex-
actly complementary template-dependent manner, in that
it will “read” the sequence of bases on the template
strand and then synthesize the complementary strand. The
start point for DNA polymerase on the parental strand
that is 5⬘33⬘ orientation to begin with is a short segment
of an RNA primer (Okazaki fragment), which is laid
down complementary to the DNA template by an enzyme
known as RNA polymerase or Primase. Because DNA
synthesis can only occur 5⬘ to 3⬘, a molecule of a second
type of DNA polymerase, ⑀ (epsilon) in eukaryotes, binds
to the other template strand, called the lagging strand as
the double helix opens. The other strand grows in the
opposite direction because it is complementary and not
identical to the template strand. A free 3⬘OH group is
required for replication, but when the two chains separate,
no group of that nature exists. The replication fork moves
in one direction, but DNA replication only goes in the 5⬘
to 3⬘ direction. This paradox is resolved by the use of
Okazaki fragments. These are short, discontinuous repli-
cation products that are produced off the lagging strand.
RNA primers are synthesized, and the free 3⬘OH of the
primer is used to begin replication. This is in comparison
to the continuous strand that is made off the leading
strand. Hence, at any given point there are two DNA
polymerases that are active at the Y-shaped junction of
the replication fork, one on the leading strand adding nu-
cleotides to make the leading strand and the other poly-
merase lays down the RNA primers for the lagging
strand. The final product does not have RNA stretches in
it. The Okazaki fragments are removed by the action of
an enzyme called RNAseH and the gaps that result from
the removal of the RNA primer are filled in by the action
of DNA polymerase I and joined together by a final bond
added by an enzyme called DNA ligase.
CONTROL OF DNA REPLICATION
Accurate and complete DNA replication is essential for
genome stability in all organisms. In eukaryotes, the DNA
is normally replicated only once during the S phase of
cell cycle. Because the replication is initiated simulta-
neously on multiple replication origins along each chro-
mosome, strict control is required for origin activation
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and density. With their multiple origins, how does the
eukaryotic cell know which origins have been already
replicated and which still await replication? Not until mi-
tosis is completed, can freshly synthesized DNA be repli-
cated again. Studies in various eukaryotes have defined a
conserved pathway of cell-cycle regulated proteins (20
different proteins) assembly and disassembly at DNA rep-
lication origins. These include the MCM (minichromo-
some maintenance) proteins, MCM2-7. These proteins
interact with each other and all six of them are required
for both initiation and elongation of DNA replication.
These are loaded during late mitosis and the G1 phase of
cell cycle; this process is called replication “licensing.”
To be replicated, each origin of replication must be bound
by an origin recognition complex of proteins (ORC).
These remain on the DNA throughout the process. Acces-
sory proteins called licensing factors accumulate in the
nucleus during G1 of the cell cycle. They include CDC6
and CDT1, which bind to the ORC and are essential for
coating the DNA with MCM proteins. Only DNA coated
with MCM proteins can be replicated. Once replication
begins in S phase, CDT1 and CDC6 leave the ORCs. The
MCM proteins leave in front of the advancing replication
fork, and reloading of MCM proteins is inhibited by at
least two proteins, geminin and the cyclin-dependent ki-
nases. Once the DNA has replicated and two identical
copies of the chromosomes have been generated, the cell
divides and the new chromosomes are segregated equally
between the two daughter cells as the cell divides.
CONCLUSION
We have provided here a brief introduction to DNA
and this by no means is meant to be an exhaustive trea-
tise on this topic. This was structured so that a novice
could read this at one go and get reasonably well updated
on the topic without falling asleep. The reader is re-
quested to refer to the excellent books and review articles
for additional reading material (4 – 8). Furthermore, a
small glossary that provides short definitions of some of
the terms italicized in the text is included. We have pro-
vided references to on-line links, such as the National
Center for Biotechnology Information (NCBI) at the Na-
tional Institute of Health which is an excellent one-stop
shop for all genome resources (9,10). It includes data-
bases such as the gene sequence database (Genbank) (9),
Online Mendelian Inheritance in Man (OMIM) (10) data-
base that provides comprehensive summary of various
genes and diseases (11,12), the literature database
 Reprinted from Academic Radiology, Vol 10, No 11, November 2003 A PRIMER ON MOLECULAR BIOLOGY FOR IMAGERS
(PubMed) (11), and several on-line books (Books central)
(12). Additionally, tools for DNA analysis, Data mining,
and links to various molecular databases are also avail-
able. The subsequent reviews in this series will focus on
the other key molecules and processes that comprise a
cell and include topics such as “Transcription and gene
expression analysis” and “Proteins and proteomics.”
REFERENCES
1. The double helix – 50 years. Nature 2003; 421(suppl):396-401.
2. Collins FS, Green ED, Guttmacher AE, Guyer MS. A vision for the fu-
ture of genomics research. Nature 2003; 422:835– 847.
3. Alberts B. DNA replication and recombination. Nature 2003; 421:431–435.
4. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD, eds. Mo-
lecular biology of the cell. 3rd ed. New York/ London: Garland Pub-
lishing, 1994.
5. Griffiths AJF, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM, eds. Intro-
duction to genetic analysis. 7th ed. New York, NY: WH Freeman, 1999.
6. Griffiths AJF, Gelbart WM, Miller JH, Lewontin RC, eds. Modern ge-
netic analysis. New York, NY: WH Freeman, 1999.
7. Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell JE,
eds. Molecular cell biology. 4th ed. New York, NY: WH Freeman, 1999.
8. Lewin B, ed. Genes VII. Oxford, UK: Oxford University Press, 2000.
9. National Center for Biotechnology Information. Genbank, gene se-
quence database. Available at: http://www.ncbi.nlm.nih.gov/Genbank/
index.html. Accessed July 15, 2003.
10. National Center for Biotechnology Information. Online Mendelian in-
heritance in man. Available at: http://www.ncbi.nlm.nih.gov/entrez/
query.fcgi?db⫽OMIM. Accessed July 15, 2003.
11. National Center for Biotechnology Information. Available at: http://
www.ncbi.nlm.nih.gov/. Accessed July 15, 2003.
12. National Center for Biotechnology Information. Available at: http://
www.ncbi.nlm.nih.gov/entrez/query.fcgi?db⫽Books. Accessed July 15,
2003.
GLOSSARY
Cell cycle: A highly ordered process that involves
DNA replication followed by cell division. It has four
main phases, the G1, S, G2, and M phase. G1, or the gap
phase, is the longest during which the cells prepare for
replication. S phase, or DNA synthesis phase, is when the
cell replicates the DNA and makes a complete copy of
the chromosomes. G2, or the second short gap phase, is
before the cell undergoes mitosis or M phase, during
which the new chromosomes are segregated into the two
daughter cell and the cell divides.
DNA Helicases: Proteins that bind to the double-
stranded DNA and stimulate the separation of the two
complementary strands.
DNA Ligase: Following the removal of Okazaki frag-
ments on the lagging strand, nicks occur in the develop-
ing DNA strand molecule because the RNA primer is
removed and synthesis proceeds in a discontinuous man-
ner on the lagging strand. These nicks are absent in the
final replication product because DNA ligase forms a co-
valent phosphor-diester linkage between 3⬘-hydroxyl and
5⬘-phosphate groups.
DNA Polymerases: A DNA-dependant DNA polymerase
links individual nucleotides together into a long strand using
another strand as a template; the product is a complementary
DNA strand. There are several subtypes of DNA poly-
merases such as ␣␮ , ␤␮, ␥, and ␦ in eukaryotes␮.
DNA single-stranded binding proteins (SSB): Special-
ized proteins that bind to the DNA as a tetramer and sta-
bilize the single-stranded DNA strands that are generated
by the action of the helicases.
Functional genomics: The study of all aspects of the
systematic analysis of gene function in an organism, cov-
ering studies of complex and model organisms dealing
with the post-sequencing phases of genome analysis.
Genome: The sum of all the genetic information in an
organism.
Genomics: The science of mapping and sequencing the
genome of an organism that is the identification and cata-
loging of all gene sequences.
Homologous: The relationship said to exist between
two genes when it is clear from their sequence that they
are evolutionarily related. This means that their sequences
share a high degree of homology and are similar, but it
does not necessarily mean that the functions of the gene
products are identical.
Metabolomics: The term “metabolome” refers to the
entire complement of all the small molecular weight metabo-
lites inside a cell suspension (or other sample) of interest.
Metabolomics is the large-scale study of the metabolome.
Pharmacogenetics/Pharmacogenomics: The study at the
genetic level of how and why different individuals re-
spond differently to various drugs. In other words, it is
the study of individual variation in drug response and
how an individual’s genetic inheritance affects the body’s
response to drugs. The term comes from the words phar-
macology and genomics and is thus the intersection of
pharmaceuticals and genetics.
Phenomics: The investigation of the “phenome” which
is the complete phenotypic representation of an organism
collected systematically and the development of new
methods to analyze such phenotypic data.
Primase: The enzyme that synthesizes the RNA primer
(Okazaki fragments) required on the lagging strand to
generate a free 3⬘ hydroxyl group at the initiation sites for
DNA replication.
Proteomics: The science of global analysis of protein
expression to identify, quantify and characterize, and cata-
logue all proteins in an organism.
S15

A Primer on Molecular Biology for Imagers:
II. Transcription and Gene Expression1
We hope the readers enjoyed the first review (1) in this
series and the introduction to DNA, structure, and func-
tion. Continuing the series, in the next two articles we
will address the central dogma in molecular biology,
which is
DNA 3 RNA 3 Protein
RNA molecules are synthesized from DNA templates, a
process called transcription, and proteins are synthesized
from RNA templates, a process called translation (Fig 1).
According to the central dogma, the genetic material con-
sists of DNA, which is capable of self-replication as well
as being transcribed into mRNA. The mRNA serves in
turn as the template for protein synthesis. Each of these
processes are highly complex and precise and enables the
cell to amplify the signal so that one molecule of DNA/
gene gives rise to many copies of RNA and each copy of
RNA gives rise to many copies of proteins, the net result
being a strong amplification of the signal. All of these
processes are highly regulated and the cell can rapidly
generate many copies and have a rapid turnover if a pro-
cess needs to be shut down, hence providing a tight con-
trol on the timing and level of expression.
Because DNA is located in the nucleus of eukaryotic
cells, whereas protein synthesis takes place in the cyto-
plasm, RNA plays the role as an intermediate in the flow
of genetic information from DNA. RNA molecules that
serve as templates for protein synthesis are called messen-
Reprinted with permission from Acad Radiol 2004; 11:333–344
1 From the Molecular Imaging Laboratory, Department of Diagnostic Radiol-
ogy, Clinical Center, 10/1N306, National Institute of Health, 9000 Rockville
Pike, Bethesda, MD 20892 (S.D.P., K.C.P.L.). Received and accepted October
21, 2003. Address correspondence to S.D.P. e-mail: spandit@mail.cc.nih.gov
© AUR, 2004
doi:10.1016/j.acra.2004.10.003
S16
Chapter II
Sunil D. Pandit, PhD, King C.P. Li, MD, MBA
ger RNAs (mRNAs). RNA and DNA also differ in size
and structure, in that RNA molecules are shorter than
DNA molecules, and that RNA is single-stranded, not
double-stranded like DNA, its sugar component is ribose
instead of deoxyribose, and it contains the pyrimidine
base uracil (U) instead of thymine (T). RNA is primarily
located in the cytoplasm and the substitution of U for a T
does not alter base pairing with an A residue. Because of
its single-stranded nature, RNA is very sensitive to degra-
dation. Hence, molecular biologists have to be extremely
careful when handling RNA in the laboratory. Function-
ally, DNA is responsible for the storing and transmission
of genetic information whereas RNA has many roles de-
pending on the type.
For a while the biologists believed that RNA was the
basic hereditary material and the key genetic component
inside the cell, until the experiments performed by Os-
wald Avery, Colin McLeod, and Maclyn McCarty in
1944 proved that DNA was the hereditary material. RNA
genomes were first discovered in plant viruses, many of
which were found to be composed of only RNA and pro-
tein. Retroviruses such as human immunodeficiency virus
(HIV) have double-stranded RNA as their genome and
use enzymes such as reverse transcriptase encoded in
their genome to convert the RNA to DNA and the DNA
subsequently integrates into the cellular DNA the viruses
infect. Howard Temin and David Baltimore independently
in 1970 discovered that the RNA tumor viruses contain
the enzyme called reverse transcriptase that catalyzes the
synthesis of DNA from an RNA template (reverse tran-
scription). The use of reverse transcriptase has enabled
mRNAs of eukaryotic cells to be converted to cDNA
(complementary DNA), which have been extensively se-
quenced as a part of human genomes expressed sequence
tags sequencing project. There are different types of RNA
molecules inside the cell classified based on the role they
play.
Reprinted from Academic Radiology, Vol 11, No 3, March 2004 TRANSCRIPTION AND GENE EXPRESSION

Figure 1. The central dogma. Gene expression occurs in two steps. In the first step,
information encoded in DNA is transcribed into a molecule of RNA and in the subse-
quent step information encoded in the mRNA is translated into a defined sequence of
amino acids.

Figure 2. Transcription by E. coli RNA polymerase. The polymerase initially binds non-
specifically to DNA and migrates along the molecule until the ␴ subunit binds to the
⫺35 and ⫺10 promoter elements, forming a closed-promoter complex. The polymerase
then unwinds DNA around the initiation site, and transcription is initiated by the poly-
merization of free NTPs. The ␴ subunit then dissociates from the core polymerase,
which migrates along the DNA and elongates the growing RNA chain. Copyright ©
2000 from Cooper GM. The Cell: A Molecular Approach. 2nd ed. Reproduced by per-
mission of Sinauer Associates, Inc.

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 PANDIT AND LI
TYPES OF RNA
Ribosomal RNA (rRNA)
Ribosomal RNA exists outside the nucleus in the cyto-
plasm of a cell in structures called ribosomes, which are
small, granular structures and the machinery for synthe-
sizing proteins by translating mRNA. There are four
kinds of rRNA, 28S (approximately 4,800 nt; S for Sved-
berg unit), 18S (approximately 1,900 nt), 5.8S (approxi-
mately160 nt) and 5S (approximately 145 nt) in eu-
karyotes. In prokaryotes, these are 23S, 16S, and 5S
rRNA which are 2,904, 1,542 and 120 nucleotides in
length, respectively. Each ribosome is a complex consist-
ing of about 60% ribosomal RNA and 40% protein. The
18S rRNA along with about 30 different protein mole-
cules form the small subunit of the ribosome. One each
of these molecules the 28S, 5.8S, and 5S rRNA, along
with some 45 different proteins, comprise the large sub-
unit of the ribosome. As we will discuss later, the 28S,
18S, and 5.8S molecules are produced by the processing
of a single primary transcript from a cluster of identical
copies of a single gene. The 5S molecules are produced
from a different cluster of identical genes.
Messenger RNA (mRNA)
Messenger RNAs are the nucleic acids that carry infor-
mation from DNA in the cell nucleus to the ribosomes in
the cytoplasm. Messenger RNA comes in a wide range of
sizes reflecting the size of the polypeptide it encodes.
Most cells produce small amounts of thousands of differ-
ent mRNA molecules, each to be translated into a peptide
needed by the cell. The mRNA encoding for housekeep-
ing proteins such as enzymes for glycolysis are expressed
in all cell types. On the other hand, there are cell-type
specific mRNAs such as the hepatocyte nuclear factor that
are liver transcription factors that moderate and coordi-
nate the expression of number of hepatocyte specific
genes. On average, a total of 10,000 to 20,000 different
mRNA species is normally observed in each cell, most
species being present at a low level (5 to 15 molecules
per cell). Most of the total cytoplasmic RNA is rRNA,
and only 3% to 5% is mRNA, a ratio consistent with the
presence of about 10 ribosomes per mRNA molecule.
Transfer RNA (tRNA)
These are the RNA molecules that carry amino acids
to the growing polypeptide and hence the name transfer
RNA. The function of transfer RNAs (tRNA) is to deliver
amino acids one by one to protein chains growing at ribo-
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Reprinted from Academic Radiology, Vol 11, No 3, March 2004
somes. There are some 32 different kinds of tRNA in a
typical eukaryotic cell and each is the product of a sepa-
rate gene. The tRNA molecules are small in size (approx-
imately 4S) containing 73–93 nucleotides, and have ex-
tensive intra-molecular hydrogen bonding to form second-
ary structures whereas the unpaired regions form three
loops. Similar to mRNA and rRNA, the mature tRNA
molecule is also generated from processing of a pre-tran-
script. Each kind of tRNA carries at its 3⬘ end, one of the
20 amino acids and hence most amino acids have more
than one representative tRNA. At one loop, three un-
paired bases form an anticodon. Base pairing between the
anticodon and the complementary codon on mRNA mole-
cule brings the correct amino acid into the growing
polypeptide chain. Further details of this process will be
described in the subsequent review article on proteins and
translation.
Small Nuclear RNA (snRNA)
DNA transcription of the genes for mRNA, rRNA, and
tRNA produces large precursor molecules called primary
transcripts that must be processed within the nucleus to
produce the functional molecules that are exported to the
cytosol. Some of these processing steps are mediated by
snRNAs. Approximately a dozen different genes for snR-
NAs, each present in multiple copies, have been identi-
fied. Several snRNAs are part of the spliceosome that
participates in splicing, ie, conversion of pre-mRNA into
mRNA by excising the introns and splicing the exons
(will be discussed later).
Small Nucleolar RNA (snoRNA)
snoRNAs within the nucleolus have several functions
and there are about 100 of these molecules in a cell. They
are responsible for splicing of the 45S rRNA precursor to
28S, 18S, and 5.8S molecules and participate in making
ribosomes, chemically modify many of the nucleotides in
the rRNA molecules by adding methyl groups to ribose
and others serve as templates for the synthesis of telo-
meres. In vertebrates, the snoRNAs are made from introns
removed during RNA processing.
MicroRNA (miRNA), Small Temporal RNA (stRNA),
and Short Interfering RNA (siRNA)
These are tiny (approximately 22 nts) RNA molecules
that appear to regulate the expression of messenger RNA
(mRNA) molecules. During RNA interference (RNAi),
long double stranded (ds) RNA is processed to approxi-
 Reprinted from Academic Radiology, Vol 11, No 3, March 2004
mately 21 nt duplexes, siRNA, which bind to mRNA and
silence genes through a mRNA degradation pathway.
stRNAs and miRNAs are approximately 21 nt RNAs that
are processed from endogenously encoded hairpin-struc-
tured precursors and function to silence genes via transla-
tional repression.
XIST RNA
This inactivates one of the two X chromosomes in fe-
male vertebrates. Xist encodes a large, spliced, polyade-
nylated, noncoding RNA that is expressed exclusively
from the otherwise inactive X chromosome. The silencing
of the inactive X chromosome is achieved by an X chro-
mosome wide alteration in chromatin structure, from ac-
tive euchromatin to inactive heterochromatin. The Xist
gene lies within the X-inactivation center and is required
to initiate X chromosome inactivation.
TRANSCRIPTION, THE PROCESS OF
GENERATING RNA
Transcription is the enzymatic synthesis of an RNA
copy on a DNA template and is the first step in gene ex-
pression. It involves the coordinated enzymatic activity of
many different proteins, and their interactions with DNA
and RNA. The factors function by making direct contacts
with the core transcriptional apparatus and/or DNA to
influence transcription. As transcription plays a central
role in biology, defects in these regulatory processes usu-
ally have severe consequences for the organism. Regula-
tion of transcription is the key step in most processes of
adaptation and differentiation, and hundreds of transcrip-
tion factors, necessary for expression of specific genes
and control of transcription elongation and termination,
have been identified. In most mammalian cells, only 1%
of the DNA sequence is copied into a functional RNA
(mRNA). Only a part of the DNA is transcribed to pro-
duce nuclear RNA, and only a minor portion of the nu-
clear RNA survives the RNA processing steps described
in the subsequent sections.
The process of transcription is similar to that of DNA
replication described previously (1). The DNA double
helix has to unwind and the bases on the two strands
have to be exposed, but only one strand is transcribed to
mRNA whereas both strands are copied during DNA rep-
lication. The DNA strand that is transcribed is called the
template strand or anti-sense strand and the sequence of
the mRNA is a copy of the complementary strand of
DNA also called as coding strand or sense strand (Fig.
TRANSCRIPTION AND GENE EXPRESSION
1). Several protein transcription factors bind to promoter
sites, usually on the 5⬘ side of the gene to be transcribed.
An enzyme, an RNA polymerase, binds to the complex of
transcription factors. Working together, they open the
DNA double helix. In a DNA molecule, either strand may
serve as the template for transcription, but the RNA poly-
merase proceeds along a strand in 3⬘ 3 5⬘ direction only.
RNA nucleotides complementary to the bases on the tem-
plate strand are assembled in the proper order by hydro-
gen-bonding, using the rules of base pairing to their com-
plementary bases on DNA and these nucleotides are
joined together by a DNA-dependent RNA polymerase
enzyme to produce mRNA (RNA polymerase II specifi-
cally for mRNA). Synthesis of the RNA proceeds in the
5⬘ 3 3⬘ direction. The mRNA produced by transcription
is a copy of the DNA coding strand. Thus for each “C”
encountered on the DNA strand, a “G” is inserted in the
RNA, for each “G” a “C” and for each “T,” an “A.”
However, each “A” on the DNA guides the insertion of
“U.” When the RNA polymerase encounters a termination
signal called terminator (in prokaryotes), which is a spe-
cific sequence of nucleotides, the polymerase and the
transcript are released from the DNA.
THE PLAYERS IN TRANSCRIPTION
The RNA Polymerases
The RNA polymerases (RNAP) are huge multi-subunit
protein complexes. There are three kinds of RNA poly-
merases in eukaryotes.
1. RNA polymerase I (RNAP I). It transcribes the 45S
rRNA precursor from rRNA genes, the precursor is
subsequently spliced to form the 28S, 18S, and 5.8S
molecules. The 45S pre-rRNA is synthesized by the
RNAP I in the nucleolus.
2. RNA polymerase II (RNAP II). This enzyme is lo-
cated in the nucleoplasm and transcribes the mRNA
and snRNA genes.
3. RNA polymerase III (RNAP III). It transcribes the
precursor for 5S rRNA, tRNA and other small nu-
clear and cytosolic RNAs. The enzyme has 16 or
more subunits and is located in the nucleoplasm.
In Escherichia coli, the core enzyme consists of five
subunits: two ␣, one ␤, one ␤⬘, and one ␻. ⬃The com-
plete enzyme, also known as holoenzyme, contains an
additional ␴ subunit. The complete enzyme is required for
transcription initiation, whereas the ␴ subunit is relatively
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 PANDIT AND LI
weakly bound and dissociates from the core enzyme re-
sponsible for transcriptional elongation. The two ␣ sub-
units are involved in promoter binding. The ␤ subunit
may be involved in both transcription initiation and elon-
gation. The ␤⬘ subunit is involved in template DNA bind-
ing. Amino acid sequence similarities between the ␤⬘sub-
unit of E. coli RNA polymerase, which binds to DNA
and the largest subunit of eukaryotic RNA polymerase II,
are among the comparisons that show a common evolu-
tionary origin for the bacterial and eukaryotic enzymes.
DNA Elements
In eukaryotes, the process of transcription is complex
and it has been a challenge to understand how transcrip-
tional control in eukaryotes is achieved by regulating hun-
dreds of thousands of promoters to yield desired levels of
mRNA. It is now clear that promoters are governed both
by the number and type of promoter-proximal and en-
hancer elements and by the action of regulatory proteins
that recognize these elements.
Promoters—The process of transcription begins by the
binding of RNA polymerase to specific DNA sequences
called promoters to initiate RNA synthesis. These se-
quences on the DNA are located upstream of the tran-
scription start site. The sequence elements or promoters
are often conserved between different genes and these
regions are sites for the binding of DNA binding proteins
or RNAP to regulate transcription. On the other hand, the
differences in promoter sequences of different genes give
rise to the variation in the levels of gene expression and
regulation. One important class of core or minimal pro-
moters in eukaryotes only consists of TATA-box, which
directs transcriptional initiation about 30 bp downstream
(Fig. 4). The TATA-box consensus is TATAAAA, which
is conserved in most eukaryotes although several mis-
matches to the consensus are allowed. Another class of
minimal promoters do not contain TATA-box and are
therefore referred to as TATA-less promoters. In these
promoters, the exact position of transcriptional start is
controlled by another basic element called initiator (INX).
The consensus sequence for initiator is loose, PyPyAN-
(TA)PyPy, and it surrounds the transcription start site
which is usually the first “A.” Another promoter element
called downstream promoter element (DPE) found in
TATA-less, initiator promoters is located 30 bp down-
stream of the transcription start site and may function as
TATA box and assist initiator containing promoters in
precise transcription initiation. The core promoter is about
60 bp in length spanning the transcription start site.
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Reprinted from Academic Radiology, Vol 11, No 3, March 2004
Table 1
DNA Binding Motifs of Transcription Factors
Consensus DNA
Transcription Factors Binding Sequence
Activator protein 1 (AP1) TGACTCA
Activator protein 2 (AP2) CCTGGGG A
CCAAT/Enhancer binding protein (C/EBP) CCAAT
E-box binding proteins (E12, E47, E2-2) CANNTG
GC2 CCGCCC
Octamer binding proteins (OCT-1 and
OCT-2) ATGCAAAT
Specificity protein 1 (Sp1) GGGCGG
Sequences of E. coli promoters are characterized by
two sets of sequence elements located 10 and 35 base
pairs upstream of the transcription start site (⫹1). The
⫺10 consensus sequences is TATAAT and is also called
the Pribnow box. The ⫺35 sequence has a consensus 6
bp sequence, TTGACA. These sequences correspond to
the bases most frequently found in different promoters.
The transcription start site is almost always a purine with
“G” being more common than “A” as the transcription
start site nucleotide.
Upstream regulatory sequences, enhancer and
repressor elements—To sustain transcription in vivo in
eukaryotes, a core promoter needs additional short regula-
tory sequences, which are located at varying distances
from the transcriptional start. Some regulatory elements
are adjacent to the core promoter and are called proximal
elements, while other elements are positioned several ki-
lobases, upstream or downstream of the promoter and are
called enhancers. Both types of elements are binding sites
for transcription factors (proteins) that increase the level
of transcription from core promoters and lead to activated
transcription. Two common elements located 100 –200 bp
upstream of the promoters with or without TATA box are
SP1 box sequence and the CCAAT box (Table 1). Simi-
larly, DNA elements that lead to reduction in transcrip-
tion levels also exist and are called repressor sequences.
A typical eukaryotic gene contains several enhancers that
are responsible for a subset of the overall gene expression
pattern. Each enhancer is about 500 bp in length and con-
tains binding sites for several sequence specific transcrip-
tion factors including activators and repressors. Binding
of sequence specific transcriptional activators to distal
enhancers leads to formation of multi-subunit transcrip-
tion complexes through protein-protein interactions. Tran-
scriptional activation is achieved by the recruitment of the
 Reprinted from Academic Radiology, Vol 11, No 3, March 2004
basal transcription machinery, ie, RNAP II to the core
promoter (described below) through protein-protein inter-
actions, either directly or through adaptor proteins.
Eukaryotic Transcription Factors
Transcription factors usually have two or three do-
mains: the transcriptional activation domain, the DNA-
binding domain, and a dimerization domain. Most eukary-
otic transcription factors that have been studied exten-
sively are activators, which stimulate transcription.
Transcriptional activators are currently thought to consist
of 40 – 80 amino acid tracts that promote target gene ex-
pression through multiple contacts with proteins in the
transcriptional apparatus. Transcriptional activators also
have effect on local chromatin structure by modifying
histone or recruiting other protein complexes which have
histone modifying activities. However, proteins that re-
press transcription have also been identified in eukaryotes.
Some repressor proteins function by binding to DNA se-
quences that overlap activator-binding sites. Other repres-
sors function by binding to sequences that overlap a tran-
scription start site, much like prokaryotic repressors. In
both cases, binding of a repressor molecule to a specific
DNA site blocks binding of proteins required to initiate
transcription. Other repressors may mask the activation
surface of an activator or interact with the general tran-
scription factors. GAL80 in yeast masks the activation
domain of the transcription factor GAL4. In some cases,
eukaryotic repressors inhibit transcription without interfer-
ing with the binding of an activator or general transcrip-
tion factor. One important example is the protein encoded
by the Wilm’s tumor (WT1) gene, which is expressed
preferentially in the developing kidney. Children that in-
herit mutations in both the maternal and paternal WT1
genes and produce no functional WT1 protein, invariably
develop kidney tumors early in life. The WT1 protein has
a zinc-finger DNA-binding domain, and binding sites for
the protein were discovered in the control region of the
gene encoding a transcription activator called EGR-1.
WT1 represses transcription of the EGR-1 promoter re-
gion and appears to be the functional converse of activa-
tors.
In E. coli, the cyclic AMP receptor protein (CRP) is a
global transcription regulator that is involved in the con-
trol of more than 100 genes. It is able to activate tran-
scription from sites both within and upstream of the re-
gion of DNA bound by RNA polymerase.
TRANSCRIPTION AND GENE EXPRESSION
Common domains in transcription factors
DNA-binding domains.—Zinc finger domains consist of
loops in which a ␣ helix and a ␤ sheet coordinately bind a
zinc ion. This domain exists in two forms. The C2H2 zinc
finger has a loop of 12 amino acids anchored by two cys-
teine and two histidine residues that tetrahedrally coordinate
a zinc ion. Usually three or more C2H2 motifs are required
for DNA binding. SP1 and the RNAP III transcription factor
TFIIIA are good examples of factors with this domain. A
related motif common to steroid hormone receptor transcrip-
tion complex is the zinc finger that contains four cysteine
amino acids, C4 that coordinate zinc ion.
Helix-turn-helix domain is one of the oldest and most
studied domains characteristic of DNA binding proteins.
It consists of 180 nucleotide sequence called homeobox
that encodes for 60 amino acid homeodomain. Common
examples of proteins that contain this domain include
bacteriophage ␭ cro repressor, lac and trp repressors, and
the antennapedia factor from drosophila. The latter consist
of three or four helical regions, of which one helix (helix
3) makes most of the contacts with DNA, while helices 1
and 2 lie on top and stabilize the interaction. Develop-
mental fates along the anterior-posterior axes of animals
are controlled by clustered homeotic genes (that contain
homeodomain) which in vertebrates are called Hox genes.
The gene clusters are similar and functionally homolo-
gous in animals as different as nematodes, flies, and
mammals. Pax (paired box) factors are also a highly con-
served family of transcription factors belonging to the
helix turn helix class. In higher vertebrates, nine members
of the Pax family have been isolated which are classified
into four paralog groups. The expression of Pax genes is
temporally and spatially restricted during development of
CNS and various other organs. Their key role in cell fate,
early patterning, and organogenesis indicates that Pax
genes are master control genes.
A basic domain is found in a number of DNA-binding
proteins and is associated with leucine zipper or helix-
loop-helix motifs. The DNA-binding domains of leucine
zipper proteins are formed from two distinct polypeptide
chains. Interactions between the hydrophobic side chains
of leucine residues exposed on one side of a helical re-
gion (the leucine zipper) are responsible for dimerization.
This brings together the two basic domains adjacent to
the leucine zipper, allowing the basic domains to interact
with DNA. Helix-loop-helix domains are similar to
leucine zippers, except that the dimerization domains of
these proteins each consist of two helical regions sepa-
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 PANDIT AND LI
rated by a loop. This domain is found in the MyoD fam-
ily of proteins.
Dimerization domains.—Such as leucine zipper, and
helix-loop-helix domains are described above.
Transcription activation domains.—Acidic activation
domain present in yeast proteins GAL4, GCN4, and her-
pes virus protein VP16 has regions that have high propor-
tion of acidic amino acids. These regions are called acid
blobs or acidic activation domains.
Glutamine-rich domains have a high proportion of
amino acid glutamine that are responsible for transcription
activation. A good example is the well-known transcrip-
tion factor SP1.
Proline-rich domain has a continuous stretch of proline
amino acids that are responsible for transcription activa-
tion. C-jun and AP2 are few examples of factors contain-
ing this domain.
PROKARYOTIC TRANSCRIPTION
The RNA polymerase core enzyme in E. coli, ␣2␤␤⬘␻,
in general has a loose affinity for DNA sequences (Fig 2).
When sigma ␴ factor binds to the core polymerase to
form the holoenzyme (␣2␤␤⬘␻␴), it markedly increases
the specificity of the holoenzyme for promoter sequences.
At the promoter the polymerase recognizes the ⫺35 and
the ⫺10 DNA sequences (Pribnow box) mentioned previ-
ously and forms a closed complex with the promoter
DNA. It is sigma’s job to recognize the promoter sites
and tell the DNA dependent RNA polymerase where to
begin transcription (initiation). Once the RNA polymerase
has been directed to the start point of the gene by sigma,
the sigma factor is released and the RNA polymerase
progresses rapidly along the DNA to carry out the process
of transcription (elongation). This also allows for re-initi-
ation of transcription from the same promoter by addi-
tional RNAP holoenzymes. The most common stop signal
in prokaryotes is the RNA hairpin in which the RNA
transcript is self-complementary. This RNA hairpin is
very GC-rich and forms a stable stem structure. This hair-
pin is followed by a stretch of four or more U residues in
the transcript. When the RNAP reaches a terminator se-
quence (stop signal) at the end of the transcription unit,
the polymerase pauses at the hairpin structure and the U
residues on the RNA base pair weakly with the A resi-
dues on the DNA anti-sense strand, hence favoring the
release of RNA from the transcription unit. Not all termi-
nator sites form a strong hairpin structure and in those
cases an accessory factor called rho aids in terminating
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Reprinted from Academic Radiology, Vol 11, No 3, March 2004
the process of transcription. Rho is a hexameric protein
that hydrolyzes adenosine triphosphate (ATP) in the pres-
ence of single stranded RNA. The rho factor binds to the
mRNA and interacts with the RNA polymerase, which
causes the enzyme to fall off the DNA template strand,
thus stopping transcription.
In prokaryotes, the genes involved in a specific meta-
bolic pathway are located adjacent to each other on the
chromosome. This unit of coordinated gene expression,
regulation, and control elements is called an operon. Two
operons in prokaryotes that have been studied extensively
include the lactose (lac) operon and the tryptophan (trp)
operon. The operon allows control of prokaryotic gene
expression through a mechanism that coordinates the ac-
tivity of a number of related genes. The lac operon con-
sists of three structural genes: lacZ (beta-galactosidase),
lacY(permease), and lacA(transacetylase) (Fig 3). The
mRNA for these genes is expressed as a single transcript
(polycistronic) from a single promoter, Plac. There is an
Olac site (operator) which lies between the Plac and the
lacZYA sites. The lacI gene encodes for a repressor
which binds as a tetramer to the operator in absence of
lactose to inhibit transcription. Lactose induces expression
of the operon by binding to the repressor and prevents the
repressor from binding to the operator.
EUKARYOTIC TRANSCRIPTION
In some aspects, transcriptional control in eukaryotes is
similar to that found in prokaryotes; namely, trans-acting
factors recognize cis-acting sites in both cases. Layered
on top of these similarities are differences imposed by the
natures of unicellular versus multicellular life, such as the
coordination of appropriate gene function in different tis-
sues. As described previously, the eukaryotes have three
different types of RNA polymerases that are responsible
for transcribing different subsets of genes. We will focus
our discussion on RNA polymerase II which transcribes
the protein coding genes and generates mRNA. The eu-
karyotic RNAP II is not capable of specific transcription
initiation in vitro but needs to be supplemented with a set
of general transcription factors (Fig 4). It is estimated
from the sequencing of the human genome that there are
about 3,000 transcription factors in humans. Regulatory
regions (described below), controlling the transcription of
eukaryotic genes, typically contain binding sites for sev-
eral transcription factors, which assemble on the basal
promoter in a specific order and affect the rate at which
RNA polymerases transcribe genes in cells. The most
 Reprinted from Academic Radiology, Vol 11, No 3, March 2004 TRANSCRIPTION AND GENE EXPRESSION

Figure 3. The lac operon. Structure of the lac operon and function of the lac
repressor.

important of the general transcription factors are TFIIA,
TFIIB, TFIID, TFIIE, TFIIF, TFIIH, each of which con-
sists of multiple subunits. The binding of RNAP II is de-
pendent on the multi-subunit complex TFIID, which is
composed of TATA-binding protein (TBP) and at least
eight TBP-associated factors. The binding of the TBP
polypeptide of the TFIID protein complex to TATA box
is the earliest step in the formation of a RNAP II tran-
scription initiation complex. TBP is present in the pre-
initiation complexes with all three RNA polymerases. The
binding of TFIID to TATA box is stabilized by TFIIA
which binds to TFIID. TFIIA is made up of at least three
subunits and appears to counteract the affects of inhibi-
tory factors such as DR1 and DR2 that are associated
with TFIID. Once TFIID has bound to the DNA, another
factor TFIIB binds to TFIID. TFIIB recruits the RNA
polymerase along with another factor TFIIF. Following
that, three other transcription factors that are required for
transcription, TFIIE, TFIIH, and TFIIJ, bind to the com-
plex. TFIIH is a large multisubunit protein complex and
contains both kinase and a helicase (DNA unwinding)
activity. The carboxyl terminus of RNAP II contains a

Figure 4. Eukaryotic promoter elements and transcription factors. The core promoter
elements consist of TATA box, initiator sequence (Inr) and downstream promoter ele-
ments. The eukaryotic transcription apparatus include multi-subunit ensembles that in-
clude RNA polymerase II core complex and associated general transcription factors.
Proximal promoter sequences and enhancers further regulate the expression from the
core promoter.

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 PANDIT AND LI
stretch of seven amino acids repeats, Tyr-Ser-Pro-Thr-
Ser-Pro-Ser, which is called carboxyl-terminal domain
(CTP). The carboxyl-terminal domain is phosphorylated
at serine and tyrosine residues by the kinase activity of
TFIID. This results in the formation of processive RNA
polymerase complex, which leaves the promoter region
and proceeds with transcription elongation.
Eukaryotic Transcription Termination Signals
The transcription termination of RNAP II transcripts
occurs by the cleavage and polyadenylation of the tran-
script rather than actual termination to generate the 3⬘ end
of mature mRNA. The tract of “A” residues is added by a
template-independent enzyme called poly (A) polymerase
(PAP) after the gene has been transcribed into pre-
mRNA. About 10 –30 nucleotides upstream of the actual
poly (A) addition site appears a consensus sequence
AAUAAA (or a slight variant) also known as the polyad-
enylation signal. It is not clear what DNA sequences sig-
nal the transcription termination but it is not the polyade-
nylation sequence. The RNAP II continues to transcribe
the pre-mRNA beyond the poly (A) addition sequence.
The nascent transcript is then cut by specific endonucle-
ase, which recognizes the poly (A) addition sequence and
creates the specific 3⬘ end for addition of poly (A) tail.
POST TRANSCRIPTIONAL PROCESSING OF
THE RNA
Splicing
Most eukaryotic genes are split into segments, exons
and introns. In general, introns tend to be much longer
than exons. In 1990, Tom Cech and Sidney Altman
shared the noble prize for their work on the mechanism
of RNA splicing. In the cell nucleus, the DNA that in-
cludes all the exons and introns of the gene is first tran-
scribed into a complementary RNA copy. In the second
step, introns are removed by a process called RNA splic-
ing to give rise to the mature transcript.
The cutting and splicing of mRNA is performed with
high degree of accuracy because if an error is made and
an extra nucleotide is removed or left behind, it can cause
a complete shift in open reading frame, ie, the reading of
the triplet code by ribosomes. The reading frame once
shifted produce new codons specifying a totally different
sequence of amino acids from that point to the end of the
molecule, or could potentially insert a stop codon leading
to premature termination of the synthesis of the peptide.
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Reprinted from Academic Radiology, Vol 11, No 3, March 2004
Under different conditions or in different cell types the
splicing of pre-mRNA for many proteins proceeds along
various paths giving rise to alternate transcripts and giv-
ing rise to a related but not identical protein in that tissue.
This process of switching to an alternate splicing pathway
is very tightly regulated to ensure that the correct tran-
script is generated. Alternative splicing provides a mecha-
nism for producing a wide variety of proteins from a
small number of genes. So, whether a particular segment
of RNA will be retained as an exon or excised as an in-
tron can vary under different circumstances.
Processing of Pre-mRNA to mRNA
The following steps elucidate the processing of pre-
mRNA and generation of a mature mRNA transcript in
eukaryotes (Fig 5). In the first step, the primary transcript
or pre-mRNA is generated in the nucleus by the action of
RNAP II transcribing the antisense strand. This pre-
mRNA has all the introns and exons. Subsequently, a
modified guanine namely 7-methylguanosine, also known
as cap is attached to the 5⬘ end of the pre-mRNA as it
emerges from RNA polymerase II (RNAP II). The cap
protects the RNA from being degraded by enzymes that
degrade RNA from the 5⬘ end. As described previously,
once the transcription is complete, the transcript is cut at
a site and the poly (A) tail is attached to the exposed 3⬘
end by the poly (A) polymerase. Following that, there is a
step-by-step removal of introns present in the pre-mRNA
and splicing of the remaining exons. This step is required
because most eukaryotic genes are split. It takes place as
the pre-mRNA continues to emerge from RNAP II. The
removal of introns and splicing of exons is done by spli-
ceosome, a complex of several snRNA molecules and
some 145 different proteins (snRNPs-small nuclear ribo-
nucleoproteins). There are specific sequences in the in-
tronic region that guide the splicesome to determine the
exact nucleotide to splice (Fig 6). The introns in most
pre-mRNAs begin with nucleotides “GU” and end with
nucleotides “AG.” The “AG” at 3⬘ end is preceded by a
pyrimidine rich sequence called polypyrimidine tract. Ten
to 40 nucleotides upstream of the polypyrimidine tract is
a consensus sequence “CURAY” (R ⫽ purine, Y ⫽ py-
rimidine) called branchpoint sequence. Splicing of mRNA
introns takes place in two steps. The 2⬘-OH of the “A”
residue in branchpoint attacks the “G” residue at the 5⬘
end of splice site to form a lariat and free exon 1. In the
subsequent step, cleavage at the “G” residue at 3⬘ end of
the intron brings the two exons together and the intron is
released in the lariat form. This generates the mature
 Reprinted from Academic Radiology, Vol 11, No 3, March 2004 TRANSCRIPTION AND GENE EXPRESSION

Figure 5. Generation of mature mRNA molecule. Transcription of anti-sense of DNA by RNA polymerase II holoenzyme generates pre-
mRNA. The nascent transcript emerging from the polymerase undergoes capping at the 5⬘ end. The 3⬘ end is poly-adenylated, following
which the pre-mRNA undergoes splicing and removal of introns. The mature mRNA molecule is now exported to the cytoplasm for
translation.

Figure 6. Mechanism of splicing of pre-mRNA. Consensus splicing signals (nucleotide

sequences) have been identified by comparing the boundaries of large number of eu-
karyotic genes. The intron is spliced out as a lariat and eventually degraded and the
two exons are joined together. Y, indicates a pyrimidine; R, a purine; N, any base; Yn,
region of about 9 pyrimidines.

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 PANDIT AND LI Reprinted from Academic Radiology, Vol 11, No 3, March 2004

Figure 7. rRNA processing. The 45S precursor of rRNA is spliced to generate 28S,
18S, and 5.8S rRNA molecules.

mRNA molecule, which is now ready for export to the
cytosol.
Processing of rRNA precursor
Human cells contain five clusters of around 40 copies
of the rRNA gene located on different chromosomes.
These genes are transcribed by RNAP I in the nucleolus.
Each rRNA gene produces a 45S rRNA precursor tran-
script which is about 13000 bases long (Fig 7). This tran-
script is processed by the splicing machinery to generate
a copy of 28S (approximately 4,718 nt) rRNA, 18S
(1,874 nt) rRNA and 5.8S (158 nt) rRNA. The continuous
transcription of multiple copies of rRNA is essential to
maintain the sufficient production of processed rRNA to
be packaged into the ribosomes.
DIFFERENTIAL GENE EXPRESSION
ANALYSIS
Clearly, the regulation of gene expression determines
the type and amount of RNA expressed inside a cell,
which determines the cell type and the organism. Gene
expression analysis in different diseases when compared
with normal tissue can define genes and pathways differ-
entially expressed in the disease state. Numerous methods
have been developed to detect and quantitate changes in
gene expression levels, including Northern analysis, dif-
ferential display, sequencing of cDNA libraries, and serial
analysis of gene expression. There are also two microar-
ray based technologies, cDNA and oligonucleotide arrays,
which enable the large scale comparative gene expression
analysis. Arrays with the capabilities to monitor the ex-
pression levels of the entire set of human genes are cur-
rently available. We will discuss these technologies in our
subsequent reviews.
SUMMARY
The process of gene expression is complex and highly
regulated to ensure that the right gene is expressed at the
right place, at the right time, and in regulated amounts.
The cell has multiple levels at which it controls the ex-
pression of a transcript including gene expression, alter-
nate splicing, and stability of the transcript. Alternate
splicing to generate different RNA species from a given
gene and DNA rearrangements where genes are rear-
ranged during cellular differentiation (eg, immunoglobulin
genes) are additional mechanisms used to generate diver-
sity in complex organisms. Epigenetic mechanisms such
as methylation where CpG-rich islands in the promoter
region depending on their methylation status can also
modulate gene expression. The reader is requested to re-
fer to the books, review articles, and web sites for addi-
tional information (2–13).
GLOSSARY
CAP: or 5-methylguanosine residue that is added to
the 5⬘ end of eukaryotic mRNA.
Constitutive or housekeeping genes: genes that are
expressed at the basal level in all tissue types.

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 Reprinted from Academic Radiology, Vol 11, No 3, March 2004
Operator: Region of an operon, close to the promoter
to which the repressor protein binds.
Operon: A cluster of bacterial genes under the control
of a single regulatory region.
Polycistronic: Many bacterial operons are expressed
as polycistronic messages. It refers to an RNA molecule
that encodes for more than one functional protein.
Reverse transcription: The process of generation of
complementary DNA (cDNA) molecule from mRNA us-
ing the enzyme reverse transcriptase.
Svedberg unit (S): A unit equal to 10⫺13 second used
for expressing sedimentation coefficients of macromolecules.
Designated symbol S after Swedish chemist Theodor Sved-
bert, who invented the ultracentrifuge and won the Noble
prize in chemistry in 1926 for his work on disperse systems.
REFERENCES
1. Special report from Nov. ‘03
2. Levine M, Tjian R. Transcription regulation and animal diversity. Nature
2003; 424:147–151.
TRANSCRIPTION AND GENE EXPRESSION
3. Hampsey M, Reinberg D. Tails of intrigue: phosphorylation of RNA
polymerase II mediates histone methylation. Cell 2003; 113:429 –
432.
4. Turner BM. Memorable transcription. Nat Cell Biol 2003; 5:390 –393.
5. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD, eds. Molecu-
lar Biology of the Cell. 3rd ed. New York/London: Garland Publishing,
1994
6. Griffiths AJF, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM, eds.
Introduction to Genetic Analysis. 7th ed. New York, NY: W. H. Free-
man & Co, 1999
7. Griffiths AJF, Gelbart WM, Miller JH, Lewontin RC (eds). Modern Ge-
netic Analysis. New York, NY: W. H. Freeman & Co, 1999.
8. Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell JE
(eds): Molecular Cell Biology. 4th ed. New York, NY: W. H. Freeman &
Co, 1999.
9. Lewin BGenes VII2000Oxford University Press
10. Cooper GM. The Cell: A Molecular Approach. 2nd ed. Sunderland,
MA: Sinauer Associates, Inc, 2000.
11. Transfac 6.0. Database on eukaryotic transcription factors and their
DNA binding sites. Available at: http://www.gene-regulation.com/pub/
databases.html transfac. Accessed January 6, 2004.
12. TESS: Transcription Element Search System. Software to identify tran-
scription factor binding sites. Available at: http://www.cbil.upenn.edu/
tess. Accessed January 6, 2004.
14. KEGG: Kyoto Encyclopedia of Genes and Genomes. Links to meta-
bolic pathways and other databases. Available at: http://www.
genome.ad.jp/kegg/kegg4.html. Accessed January 6, 2004.
S27

A Primer on Molecular Biology for Imagers:
In this article we will complete the discussion of the
last step in central dogma, which is the process of
translation and the synthesis of proteins. The comple-
tion of human genome sequence has defined the 35–
40K genes that exist in the genome of Homo sapiens.
As discussed previously (4,12), every cell type ex-
presses only a subset of these genes and it is this sub-
set of expressed genes that defines the phenotype of
the cell. In a given cell, approximately 100K proteins
are expressed, which is a combination of housekeeping
and cell-type specific proteins (1,2). Only 1%–2% of
the 3 billion base-pairs of human genome encode for
proteins, and the full complement of protein-coding
sequences remains to be established because predicting
protein coding sequences based on sequence alone re-
mains a challenge. The function of only a small per-
centage of the 40K genes in humans is known, whereas
the majority of the genes and the products they encode,
their function inside the cell, the pathways, and their
cross-talk are largely unknown. The ENCyclopedia Of
DNA Elements (ENCODE) project launched recently
by the National Human Genome Research Institute
(NHGRI) at the National Institute of Health (NIH)
hopes to elucidate all the protein coding genes and
non-coding regions of the genome (3). Additionally,
this project will identify functional elements such as
promoters, origin of replication, and other interesting
features in the human and model organisms DNA se-
quences.
Reprinted with permission from Acad Radiol 2004; 11:448 – 461
1 From the Molecular Imaging Laboratory, Department of Diagnostic Radiol-
ogy, Clinical Center, 10/1N306, National Institute of Health, 9000 Rockville
Pike, Bethesda, MD 20892 (S.D.P., K.C.P.L.). Received 00 Month 2003;
revision requested Month 00; received in revised form 00 Month 2003;
accepted 00 Month 2004.. Address correspondence to S.D.P.
© AUR, 2004
doi:10.1016/j.acra.2004.10.004
S28
Chapter III
III. Proteins: Structure and Function
Sunil D. Pandit, PhD, King C.P. Li, MD, MBA
Proteins are the building blocks of the human body.
The process of converting the information contained in
a DNA segment into proteins begins with the synthesis
of mRNA molecules (transcription) containing any-
where from several hundred to several thousand ribo-
nucleotides, depending on the size of the protein. This
mRNA then gets translated into a peptide. This process
again is very complex and highly regulated and the cell
performs it with a very low error rate. It is absolutely
amazing how many processes have to be coordinated
inside a tiny cell (⬃ 7 ␮m in size). We as scientists
are still trying to understand how parts of this machin-
ery work but are not in a position yet to create machin-
ery that functions so efficiently and error free.
Proteins are the most abundant organic molecules
inside a cell and account for at least 50% of its dry
weight. A number of proteins encode for enzymes that
carry out the cellular reactions. Again, all these reac-
tions are performed at neutral pH and at 37°C, whereas
it would take harsh conditions such as extremes of pH
and temperatures to carry out these same reactions in
vitro. In the 1940s, Linus Pauling and Robert Corey
elucidated the ␣-helix and ␤-sheet structures, which are
considered the 2 fundamental building blocks of all
protein secondary structures. Linus Pauling is also one
of the select few Nobel laureates to win the coveted
prize twice; the second time for Pauling was the Nobel
peace prize.
AMINO ACIDS: THE BUILDING BLOCKS OF
PROTEINS
A protein is a polymer composed of monomers
called amino acids and the chain of amino acids is re-
ferred to as a polypeptide. Amino acids contain at least
1 carboxyl group (COOH) and 1 ␣-amino (NH2) group
but differ from each other in the structure of the R
 Reprinted from Academic Radiology, Vol 11, No 4, April 2004 PROTEINS: STRUCTURE AND FUNCTION

Figure 1. The amino acids. The 3-letter and 1-letter abbreviations for each amino acid are indicated. The amino acids are
grouped into 4 categories according to the properties of their side chains: nonpolar, polar, basic, and acidic. (Reproduced with
permission from The Cell: A Molecular Approach. 2nd ed. Cooper GM. Sunderland, MA: Sinauer Associates, Inc.; 2000.)

groups or side chains. Amino acids all have the general The side chain, or R (reactive) group, can be anything
formula from a hydrogen atom (as in the amino acid glycine) to a
complex ring (as in the amino acid tryptophan). There are
20 different ␣-amino acids commonly found as building
H
blocks in proteins; 9 of these amino acids are essential for
兩
the body. In other words, our body makes only very small
H2N — C — COOH amounts of these amino acids and they have to be supple-
兩 mented in our diet. In protein molecules the amino acid
R residues are covalently linked together, united in a head-

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 PANDIT AND LI
to-tail arrangement through amide linkages called peptide
bonds, which is formed through a condensation reaction
during which 1 water molecule is removed. Some pro-
teins contain only 1 polypeptide chain whereas others
contain 2 or more.
PRIMARY, SECONDARY, TERTIARY, AND
QUATERNARY STRUCTURES OF PROTEINS
Proteins have a complex structure that has 4 levels of
organization. The linear sequence of the amino acids in a
polypeptide chain constitutes the primary structure of the
protein. Several types of weak bonds (hydrogen, electro-
static, and van der Waals), acting between atoms within a
polypeptide chain or between different polypeptide
chains, cause the protein to take on a specific shape. The
secondary structure of a protein arises from the interac-
tions of amino acids that are close together in the linear
sequence, most commonly forming a ␣-helix or a
␤-pleated structure. It is defined as the local conformation
of the structure’s backbone. Tertiary structure is the pro-
tein’s three-dimensional shape, incorporating the pleats
and helices along with the spatial disposition of its side
chains. It is produced by folding the helix or other sec-
ondary structure. In some proteins, 2 or more tertiary
structures come together to form a quaternary structure.
The quaternary association can be between different types
of polypeptides or between identical polypeptides and
constitutes a protein consisting of more than 1 amino acid
chain. Proteins with 2 or more polypeptide chains are
called as oligomeric proteins, a well-known example be-
ing hemoglobin, which consists of 2 ␣ chains and 2 ␤
chains and heme as the prosthetic group.
Proteins can be divided into 2 major classes, simple or
conjugated proteins, depending on their composition.
Simple proteins upon hydrolysis yield only amino acids
whereas conjugated proteins upon hydrolysis yield in ad-
dition other non–amino acid components, which can be
inorganic or organic (Prosthetic group). Depending on the
nature of the prosthetic group, conjugated proteins can be
classified as nucleoproteins, lipoproteins, glycoproteins,
metalloproteins, phosphoproteins and others.
Depending on the conformation, proteins can be fur-
ther classified into 2 broad categories, fibrous or globular
proteins. The fibrous proteins consist of polypeptide
chains arranged in parallel along a single axis to yield
long fibers or sheets. Fibrous proteins are physically
tough and insoluble in water or dilute salt solutions. They
are the basic structural elements in the connective tissue
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Reprinted from Academic Radiology, Vol 11, No 4, April 2004
and include proteins such as collagen (tendons), ␣-keratin
(hair, nails, skin) and elastin (connective tissue). Globular
proteins, on the other hand, are tightly folded into com-
pact spherical or globular structures. Most globular pro-
teins are soluble in aqueous solutions. Enzymes and anti-
bodies are among the most important globular proteins,
which also include proteins with transport function such
as serum albumin and hemoglobin.
THE TRANSLATION MACHINERY IN
PROKARYOTES AND EUKARYOTES
The Ribosomes
Ribosomes are compact ribo-nucleoprotein particles of
ribosomal RNA (rRNA) molecules (12) and specific ribo-
somal proteins. Each ribosome consists of 2 subunits, the
large and the small subunit. The prokaryotic ribosome is
70S (sediments at 70S) in size and is formed from a
larger subunit 50S in size and a small subunit 30S in size,
whereas the eukaryotic ribosome is larger in size (80S)
and is composed of 1 large 60S subunit and a smaller
40S subunit. The 50S subunit in prokaryotes is composed
of 31 different proteins in addition to 23S and 5S rRNA
molecules. The smaller subunit (30S) contains 21 differ-
ent proteins and the 16S rRNA. The eukaryotic 60S large
subunit is composed of 45 different proteins and 1 mole-
cule each of 28S, 5.8S and 5S rRNA, whereas the 40S
subunit contains an 18S rRNA and 30 distinct proteins.
Even though the eukaryotic ribosomes are more complex,
they carry out essentially the same function as their bacte-
rial counterparts.
tRNA Molecule
Because amino acids are structurally unrelated to the
nucleic acid bases, direct complementary pairing between
mRNA and amino acids during the incorporation of
amino acids into proteins is not possible. tRNA molecules
serve as adaptors between amino acids and mRNA during
translation and provide the twin functions of codon recog-
nition and amino acid recognition. In 1976, Aaron Klug
et al. described the detailed three-dimensional structure of
tRNA by using x-ray diffraction analysis. The tRNA mol-
ecule typically sediments at 4S, is approximately 60 –95
nucleotides in length; has a cloverleaf-like structure; and
contains a number of modified bases such as ribo-thymi-
dine, inosine, dihydrouridine, and others. The complemen-
tary base pairing between the bases of the tRNA enables
it to form a stem-loop structure (Fig. 2), which are also
 Reprinted from Academic Radiology, Vol 11, No 4, April 2004 PROTEINS: STRUCTURE AND FUNCTION

Figure 2. Phenylalanine tRNA of yeast. (a) The molecule is drawn with a cloverleaf shape to show the complementary base-pairing
(short gray bars) that occurs in the helical regions of the molecule. (b) The actual shape of the molecule, based on x-ray diffraction anal-
ysis, is shown schematically. Complementary base pairs are indicated as long gray bars. In addition, the nucleotides involved in unusual
base-pair interactions that hold different parts of the molecule together are colored red and are connected by a red line in both (a) and
(b). The pairs are numbered in (b). (c) One of the unusual base-pair interactions. Here 1 base forms hydrogen-bond interactions with 2
others; several such “base triples” help fold up this tRNA molecule. (Reproduced with permission from Molecular Biology of the Cell. 3rd
ed. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. New York and London: Garland Publishing; 1994.).

known as the arms of the tRNA. Prior to its use in pro-
tein synthesis, each tRNA is charged only with the amino
acid for which its anticodon is appropriate. The process
of charging the tRNA to form aminoacyl-tRNA is cata-
lyzed by a specific enzyme called aminoacyl-tRNA syn-
thetase. The tRNA molecule that serves as the initiator of
protein synthesis in prokaryotes is the N-formylmethio-
nyl-tRNA molecule whereas the eukaryotic initiator tRNA
does not form the N-formyl, but in both cases the first
amino acid synthesized in the polypeptide is methionine.
The triplet code is degenerate as more than 1 codon en-
codes for the same amino acid. The degeneracy is at the
third position in the triplet code (4). The wobble hypothe-
sis allows for the ability of the tRNA molecule to recog-
nize more than 1 codon by unusual base pairing (non
G-C, A-T) with the third base of a codon.
Translation Accessory Factors
The ribosome itself cannot synthesize the polypeptide
chain and requires the presence of accessory protein fac-
tors to carry out each of the steps in protein synthesis,
initiation, elongation, and termination. In prokaryotes, the
factors are abbreviated as IF for initiation factor or EF for
elongation factor. In eukaryotes these factors are called
eIF or eEF. The prokaryotic and eukaryotic factors are
summarized in Table 1.
Initiation, Elongation, and Termination of Protein
Synthesis
The information encoded in mRNA is translated by the
ribosome in the cytoplasm of the cell to produce a pro-
tein. The rate of prokaryotic translation is much faster, at
approximately 12–17 amino acids/sec, whereas the eu-

Table 1
Translation Accessory Factors Required in Initiation, Elongation and Termination of Protein Synthesis in Prokaryotes and Eukaryotes

Role Prokaryotes Eukaryotes

Initiation IF-1, IF-2, IF-3 eIF-1, eIF-1A, eIF-2, eIF-2B, eIF-3, eIF-4A, eIF-4B, eIF-4E, eIF-4G, eIF-5
Elongation EF-Tu, EF-Ts, EF-G eEF-1␣, eEF-1␤␥, eEF-2
Termination RF-1, RF-2, RF-3 eRF-1, eRF-3

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 PANDIT AND LI
Figure 3. Overview of translation. (Reproduced with permission from The Cell: A Molecular Approach. 2nd ed. Cooper GM. Sunderland,
MA: Sinauer Associates, Inc.; 2000.)
karyotic ribosome translates at the rate of 2 amino acids/sec.
The ribosome, along with a set of accessory factors, works
in an assembly-line fashion to carry out the initiation, elon-
gation, and termination stages of protein synthesis (Fig. 3).
The ribosome uses the universal genetic code to determine
the amino acid sequence encoded by the mRNA strand.
Only the information contained between the start (AUG) and
stop (UAA, UAG, or UGA) codons in an mRNA molecule
is used to produce an amino acid sequence. Following the
start codon (AUG), the ribosome moves along the mRNA,
reading successive triplets by admitting the appropriate ami-
noacyl-tRNA molecule that possess the corresponding anti-
codon. At each step, the polypeptide chain grows by 1
amino acid. The energy for protein synthesis is provided by
the hydrolysis of GTP. Base pairing between the anticodon
on each tRNA and the codon sequence on the mRNA then
directs the attached amino acid to its correct position on the
mRNA template.
Initiation
Protein synthesis is initiated when an mRNA, a ribo-
some, and the first tRNA molecule for methionine amino
acid come together. In prokaryotes, the ribosome is inac-
tive when it exists as 2 subunits (30S and 50S) before it
contacts an mRNA. Initiation factors 1 and 3 (IF1 and
IF3) bind to the 30S subunit and prevent the large subunit
50S from binding. This is followed by the binding of ini-
tiation factor 2 (IF2) and GTP to the smaller subunit,
which in turn facilitates the binding of initiator tRNA
(Met). This small subunit complex now attaches to the
mRNA releasing IF3. The initiation sites in prokaryotic
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Reprinted from Academic Radiology, Vol 11, No 4, April 2004
mRNAs are characterized by a Shine-Dalgarno sequence
that precedes the AUG initiation codon. Base pairing be-
tween the Shine-Delgarno sequence and a complementary
sequence near the 3⬘ terminus of 16S rRNA in the small
subunit aligns the mRNA on the ribosome. The large sub-
unit now binds to form the fully assembled 70S ribosome
initiation complex at the correct position on the mRNA.
In prokaryotes, both AUG and GUG are used as start
codons, whereas the eukaryotes use only AUG.
In contrast, eukaryotic mRNAs are bound to the 40S
ribosomal subunit by their 5⬘ 7-methylguanosine caps.
There is no Shine-Dalgarno sequence in eukaryotic
mRNA. The eukaryotic 40S small subunit of the ribo-
some will initiate the process of translation when it en-
counters an mRNA in the cytoplasm. The small subunit
complex binds to the 5⬘ cap region of the eukaryotic
mRNA and then scans along the mRNA until it encoun-
ters an AUG initiation codon. It is not the first AUG
codon on the 5⬘ end of the mRNA that acts as a “start”
signal for the translation machinery. M. Kozak proposed a
scanning model where in addition to the AUG codon
there are certain sequence requirements around the AUG
(5⬘-CCRCCAUGG-3⬘ ; R ⫽ purine), which is recognized
before translation initiates in eukaryotes. In both pro-
karyotes and eukaryotes, the assembled ribosome has 2
tRNA binding sites, in the cleft of the smaller subunit.
They are called the P and the A sites for peptidyl and
aminoacyl-tRNA sites and contain adjacent codons that
are being translated (Fig. 4). Initiation is complete when
the methionine tRNA occupies the P binding sites on the
 Reprinted from Academic Radiology, Vol 11, No 4, April 2004
Figure 4. The 3 major RNA-binding sites on a ribosome. An empty ribosome is shown on the left and a loaded ribosome on the right.
(Reproduced with permission from Molecular Biology of the Cell. 3rd ed. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. New
York and London: Garland Publishing; 1994.).
ribosome where the growing peptide chain is. This is the
only tRNA that does this, as all other tRNA molecules
have to enter the A-site first, located 3⬘ of the P-site.
Even though every protein begins with the amino acid
methionine, in some cases the methionine is cleaved of
the amino terminus in the mature protein.
Elongation
Once the initiation complex is complete, the process of
elongation of the polypeptide chain begins (Fig. 5). The
elongation process is quite similar in prokaryotes and Eu-
karyotes, where they each have an equivalent set of elon-
gation factors that carry out essentially the same function
(Table 1). The elongation process can be divided into 3
steps: the delivery of aminoacyl-tRNA, peptide bond for-
mation, and the translocation of the growing peptide. The
incoming tRNA will bind to the A-site and all available
tRNAs will approach the site and try to attach, but only
the tRNA with the anticodon sequence complementary to
the codon of the A-site on the mRNA binds. Now that
the A and the P sites are occupied, the 2 amino acids in
close proximity are joined together by the peptidyl trans-
ferase activity of the 50S subunit (prokaryotes) to form a
peptide bond. No energy (ATP) is expended at this stage.
Once the bond has been formed between the 2 amino ac-
ids, a complex of enzyme translocase and GTP binds to
the ribosome, energy is used to eject the tRNA on the
P-site, which passes its amino acid on to the tRNA on the
A-site. The peptidyl-tRNA now moves from the A-site to
the P-site and the mRNA moves by 1 codon relative to
the ribosome. The A-site is now available for the next
appropriate tRNA molecule to bring its amino acid right
PROTEINS: STRUCTURE AND FUNCTION
next to the tRNA holding the 2 amino acids. This process
repeats itself, the ribosome slides down another codon,
and this continues until the full protein is completed and
1 of the 3 termination codons appears in the A-site, in
which case the termination event occurs.
Termination
A “stop” codon (UAA, UAG, or UGA) signals the end
of the protein synthesis process. There is no tRNA that is
complementary to the stop codon. In prokaryotes there are 3
release factors (release factors 1, 2, and 3 [RF1–3]) that
cause the release of the polypeptide chain. RF1 recognizes
the codons UAA and UAG, and RF2 recognizes codons
UAA and UGA. RF3 helps RF1 and RF2 to carry out their
function. In eukaryotes there is only 1 release factor (eukary-
otic release factor, eRF) that recognizes all 3 codons. The
release factors help the peptidyl transferase to transfer the
complete polypeptide chain to a water molecule rather than
the amino-acyl tRNA present in the A-site. The mRNA mol-
ecule is released from the ribosome as the small and large
subunits fall apart. The mRNA can now either be re-trans-
lated or degraded, depending on how much of that particular
protein is needed. All mRNAs are eventually degraded once
the synthesis of the protein is no longer required.
POST-TRANSLATIONAL PROCESSING OF
PEPTIDES
Protein Translocation and Post-Translational
Modifications
In the cytosol of the cell there are 2 spatially distinct
populations of ribosomes. Some are bound to the endo-
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 PANDIT AND LI
Figure 5. The elongation phase of protein synthesis on a ribosome.
The 3-step cycle shown is repeated over and over during the synthesis
of a protein chain. An aminoacyl-tRNA molecule binds to the A-site on
the ribosome in step 1, a new peptide bond is formed in step 2, and the
ribosome moves a distance of 3 nucleotides along the mRNA chain in
step 3, ejecting an old tRNA molecule and “resetting” the ribosome so
that the next aminoacyl-tRNA molecule can bind. As indicated in Figure
4, the P-site is drawn on the left side of the ribosome, with the A-site on
the right. (Reproduced with permission from Molecular Biology of the
Cell. 3rd ed. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD.
New York and London: Garland Publishing; 1994.)
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Reprinted from Academic Radiology, Vol 11, No 4, April 2004
plasmic reticulum (ER) and others are free in the cytoplasm.
The membrane-bound and free ribosomes differ only in the
types of proteins they synthesize: the free ribosomes synthe-
size proteins that are cytosolic and all proteins that are not
destined for the ER. There are 2 distinct types of endoplas-
mic reticulum (ER): the rough ER, which is involved in pro-
tein synthesis and is covered by ribosomes on its outer sur-
face, and the smooth ER, which is not associated with ribo-
somes and is involved in lipid rather than protein
metabolism (Fig. 6). In mammalian cells, most proteins des-
tined for the endoplasmic reticulum enter the ER co-transla-
tionally. The ribosomes that are involved in the synthesis of
proteins destined for secretion are targeted to the endoplas-
mic reticulum by a signal sequence at the amino terminus of
the growing polypeptide chain (Table 2). This signal se-
quence is approximately 20 amino acids in length and con-
sists of mostly hydrophobic amino acids. A signal recogni-
tion particle (SRP) binds to the signal sequence and also the
ribosome and targets the entire complex to the endoplasmic
reticulum, which expresses the receptor for the SRP. Bind-
ing of SRP to its receptor releases SRP, the ribosome then
binds to the ER membrane, and the signal sequence is in-
serted into the membrane channel. The signal sequence is
eventually cleaved off the mature protein. Proteins destined
for incorporation into the plasma membrane or the mem-
branes of the ER, Golgi, or lysosomes are initially inserted
into the ER membrane instead of being released into the
lumen. From the ER membrane the proteins reach their final
destination following the pathway of secretory proteins,
which is the ER3 Golgi3plasma membrane or lysosomes.
This process is post-translational; the proteins are packaged
into a vesicle within the lumen of 1 organelle and then re-
leased into the lumen of the recipient organelle following
vesicle fusion. Proteins that function in ER and must be re-
tained have a short sequence at their carboxy-terminus, Lys-
Asp-Glu-Leu (KDEL, in single amino acid code), which
enables them to do so. There are other peptide sequences in
proteins that are responsible for the correct localization of
these proteins inside the cell (Table 2). Different N-terminal
sequences can cause proteins to be localized into certain
organelles, such as mitochondrial import signal, import into
peroxisomes, and the nuclear localization signal (NLS),
KKKRK (1 letter code), which consists of highly basic
amino acids and is present in proteins that are imported into
the nucleus, for example, DNA replication proteins and his-
tones.
The ER is also the site of assembly of multisubunit
proteins, disulfide bond formation, the initial stages of
glycosylation, and the addition of glycolipid anchors to
 Reprinted from Academic Radiology, Vol 11, No 4, April 2004
some plasma membrane proteins. Proteins are glycosy-
lated on specific asparagine residues (N-linked glycosyla-
tion) within the ER while their translation is still in pro-
cess. Some proteins are anchored in the plasma mem-
brane by glycolipids containing phosphatidylinositol, such
as glycosylphosphatidylinositol (GPI) anchors rather than
by membrane-spanning regions of the polypeptide chain.
The GPI anchors are assembled in the ER membrane and
added to the protein after completion of protein synthesis.
Protein Folding
Any given polypeptide is expected to have an infinite
number of different conformations that it can adapt, but it
Table 2
PROTEINS: STRUCTURE AND FUNCTION
Figure 6. The major intracellular compart-
ments of an animal cell. The cytosol (gray),
endoplasmic reticulum, Golgi apparatus, nu-
cleus, mitochondrion, endosome, lysosome,
and peroxisome are distinct compartments
isolated from the rest of the cell by at least 1
selectively permeable membrane. (Repro-
duced with permission from Molecular Biol-
ogy of the Cell. 3rd ed. Alberts B, Bray D,
Lewis J, Raff M, Roberts K, Watson JD. New
York and London: Garland Publishing; 1994.)
is only one of these native conformations that the
polypeptide actually adapts in normal biological condi-
tions of temperature and pH. Proteins acquire their func-
tional, preordained, three-dimensional structure after they
emerge as linear polymers of amino acids from the ribo-
somes. The experiments performed by Christian Anfinsen
in 1970s on ribonuclease A showed that the three-dimen-
sional structure of a protein is determined purely by the
primary structure, which is its amino acid sequence. Pro-
teins are translocated across the membrane of the endo-
plasmic reticulum (ER) as unfolded polypeptide chains
while their translation is still in progress (5,6). The pro-
cess of protein folding occurs in the ER immediately after
the protein synthesis is complete and the protein is re-
leased from the ribosome. The folding may occur as
quickly as in microseconds for small proteins to several
minutes for larger proteins, which may require several
chaperone-assisted steps to acquire its native state (7).
The formation of correct disulphide bonds, assisted by
chaperones such as protein disulphide isomerase (PDI)
occurs in the lumen of the ER, which does not have a
reducing environment like that of the cytosol. There are
many steps in the protein folding process starting with the
primary structure. This is followed by the generation of
␣-helix and ␤-sheet structures that are stabilized by hy-
drogen bonding and nitrogen– hydrogen bonding. The ter-
tiary structure is stabilized by additional bonding between
the side chains, for example, the disulphide linkages
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 PANDIT AND LI
(which are covalent bonds), which stabilize the protein.
The quaternary structure is formed by the interaction of
various multiple native proteins with a number of bonding
interactions, such as covalent bonding, to stabilize the
protein. Shape is critical to a protein because its specific
shape enables it to do its specific job in the cell. For ex-
ample an enzyme has a specific pocket, called the active
site, into which its substrate can fit. It is similar to a lock-
and-key mechanism where only 1 key can fit correctly
into the lock; that is, only the correct substrate fits into
the active site of an enzyme. The amino acid sequence
also determines which R groups are available to enable
the protein to bind to other specific cellular components.
A number of diseases are caused by a misfolded protein
(8). Alzheimer’s and Creutzfedlt-Jakob diseases are
caused by deposits of aggregated proteins in brain and
other tissues. Sickle cell anemia is another well-known
example, where the glutamic acid at position 6 in the
␤-chain of the hemoglobin is replaced by the amino acid
valine. This single amino acid substitution causes the
change in conformation of the hemoglobin-S (sickle) mol-
ecule, which differs from the normal hemoglobin-A in
such a way that the packing of the hemoglobin-S mole-
cules in the sickle erythrocyte causes it to change shape
on de-oxygenation, hence the crescent or sickle shape.
Protein Structure
It was in 1864 that Hoppe-Seyler crystallized and
named the protein hemoglobin. We all know that in 1895
Wilhelm Röentgen observed a new form of penetrating
radiation, which, was produced when cathode rays (elec-
trons) hit a metal target and which he named x-rays. Sub-
sequently, in 1912, Von Laue used x-rays to obtain the
first diffraction patterns by passing x-rays through a crys-
tal of zinc sulfide. The father-and-son team of William
Henry Bragg and William Lawrence Bragg proposed a
simple relationship between an x-ray diffraction pattern
and the arrangement of atoms in a crystal that produced
the pattern, which we all know as Bragg’s law. And so
began the field of x-ray diffraction pattern analysis. It was
not until 1934 that John Desmond Bernal and Dorothy
Crowfoot Hodgkin (the third woman ever to win the No-
bel Prize in chemistry) presented the first detailed x-ray
diffraction patterns of a protein obtained from crystals of
the enzyme pepsin. A year later, in 1935, Patterson devel-
oped an analytical method for determining inter-atomic
spacing from x-ray data. The genius Linus Pauling in
1931 had previously published his first essays on “The
Nature of the Chemical Bond,” detailing the rules of co-
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Reprinted from Academic Radiology, Vol 11, No 4, April 2004
valent bonding. But it was in the year 1951 that Pauling
and Corey studied the helical conformation of a chain of
␣ and ␤ keratins and proposed the ␣-helix and the
␤-sheet structures, both of which are commonly found in
many proteins (Fig. 7). In this structure, the ␣-helix, the
backbone is arranged in a helical coil having approxi-
mately 3.6 amino acid residues per turn. The R groups of
the amino acids extend outward from the rather tight he-
lix formed by the backbone. The naturally occurring L
amino acids can form either a right-handed or a left-
handed helix, but the right-handed helix is more stable
and hence universally present in all native proteins. In the
␣-keratins of hair and wool, 3 to 7 such ␣-helices are
coiled together around each other to form a rope-like
structure held together by disulphide bonds. The other
structure commonly seen in proteins is the ␤-pleated sheet
structure, elucidated by Pauling and Corey by studying
the ␤-keratins. Side-by-side polypeptide chains in the ␤
conformation are arranged in the form of a pleated sheet,
with the R groups above and below the planes of the
pleated sheet and the adjacent polypeptides cross-linked
by inter-chain hydrogen bonds. In 1954, Max Perutz and
colleagues developed heavy-atom methods to solve the
phase problem in protein crystallography, and later he
proposed a lower-resolution structure of the hemoglobin.
In 1971, Jean Jeener proposed the use of two-dimensional
NMR, and Kurt Wuthrich and colleagues first used the
method to solve a protein structure in the early 1980s.
NMR studies have been useful for detecting which amino
acids in a native protein molecule participate in confor-
mational changes. Because the protons of different types
of functional groups such as —OH, —CH3, etc., have
different and characteristic magnetic resonances, changes
in the local environment of such groups can be detected
by NMR measurements. Since the advent of gene cloning
in the 1970s, a number of proteins have been over-ex-
pressed in bacteria and eukaryotic systems, crystallized,
and their structures have been determined. Presently, re-
searchers are crystallizing macromolecular assemblies to
understand how such assemblies of several proteins func-
tion in vivo (9,10). The determination of the crystal struc-
ture of various proteins has enabled structure–activity re-
lationship studies and facilitated the computer-aided drug
design of small molecule inhibitors.
Protein Analysis
The ␣ amino group of the amino acids reacts with 2
molecules of ninhydrin reagent to yield an intensely col-
ored product. The ninhydrin reaction was widely used to
 Reprinted from Academic Radiology, Vol 11, No 4, April 2004 PROTEINS: STRUCTURE AND FUNCTION

Figure 7. Secondary structure of proteins. The most common types of secondary structure are the ␣-helix and the ␤-sheet. In an
␣-helix, hydrogen bonds form between CO and NH groups of peptide bonds separated by 4 amino acid residues. In a ␤-sheet, hydrogen
bonds connect 2 parts of a polypeptide chain lying side by side. The amino acid side chains are not shown. (Reproduced with permis-
sion from The Cell: A Molecular Approach. 2nd ed. Cooper GM. Sunderland, MA: Sinauer Associates, Inc.; 2000.)

generate quantitative estimates of small amounts of amino
acids. Another widely used assay to detect proteins is the
Bradford assay. It is based on the principle that the ab-
sorbance maximum following addition of an acidic solu-
tion of Coomassie Blue G-250 dye to a protein solution is
shifted from 465 nm to 595 nm. This can be measured on
a spectrophotometer or a microplate reader. The dye
binds primarily to basic and aromatic amino acids such as
arginine, tryptophan, tyrosine, histidine, and phenylala-
nine. The detection limit of this assay is 1–20 ␮g (mi-
croassay) and 20 –200␮g of protein (macro-assay). An-
other assay that is commonly used to determine protein
concentration is the Lowry assay, where the Cu⫹2 ions
under alkaline condition react with the peptide nitrogen
and the subsequent reduction of the Folin-Ciocalteay re-
agent to produce color. This reaction is sensitive to low
concentrations of protein but is subject to tight pH control
and interference by a variety of compounds.
Peptide Sequencing
A very important development was a method for iden-
tifying the N-terminus amino acid of the peptide. The first
method was developed by Fred Sanger of the Medical
Research Council, Cambridge, who used the reagent 2, 4
dinitrofluorobenzene (DNFB). This reacts with the

S37
 PANDIT AND LI
␣-amino acid at the N-terminus and forms a yellow deriv-
ative, which is cleaved using acid hydrolysis to digest all
the peptide bonds except the bond between the ␣-amino
acid and DNFB. The labeled residue can easily be sepa-
rated from the unsubstituted amino acids and identified by
chromatographic comparison with known di-nitrophenyl
derivatives of the various amino acids. Sanger used this
method to identify the complete sequence of insulin, a
di-peptide of 51 amino acids, with the 2 chains of 30 and
21 amino acids linked together by a single disulphide
linkage. It took Sanger 20 years of chromatography to
elucidate the sequence of insulin, for which he won his
first Nobel prize. He subsequently went on to develop the
method for DNA sequencing for which he won a second
Nobel Prize, which he shared with Maxam and Gilbert.
Subsequently, another method for peptide sequencing
from the N-terminus was developed by Edman, which
uses a different reagent called phenylisothiocyanate,
which reacts quantitatively with the free ␣-amino group
to form the phenylthiohydantoin derivative that can be
detected by gas–liquid chromatography (GLC). The ad-
vantage of the Edman method is that unlike Sanger’s
method, the rest of the peptide chain after removal of the
N-terminus amino acid is left intact for further cycles of
this procedure. The Edman method forms the basis of
current automated peptide sequencers and permits very
rapid determination of the amino acid sequence of pep-
tides.
Solid-Phase Synthesis
Bruce Merrifield and his colleagues developed a
method called solid-phase peptide synthesis for chemical
synthesis of polypeptides. In this procedure, the polypep-
tide chain is built amino acid by amino acid starting from
the carboxyl-terminus, which is covalently anchored to an
insoluble solid resin particle. The amino groups are pro-
tected by the tert-butyloxycarbonyl group, which is re-
moved in the form of volatile products upon acidification.
These steps are repeated and excess reagents at each step
are removed by filtration and thorough washing of the
resin particle. Once the peptide chain synthesis is com-
plete, it is cleaved from the resin and easily separated
from the solid-phase resin. This method is currently used
to synthesize peptides in vitro. In the future, one would
like to be able to synthesize complete functional proteins,
folded correctly with all the appropriate post-translational
modifications using highly efficient in vitro techniques.
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Reprinted from Academic Radiology, Vol 11, No 4, April 2004
Antibiotics and Protein Synthesis
More than 100 different antibiotics are directed against
targets that are present in bacteria but not in human cells.
Penicillin, for example, inhibits synthesis of the bacterial
cell wall. Many of these antibiotics inhibit different steps
in protein synthesis. Antibiotics such as streptomycin,
tetracycline, chloramphenicol, and erythromycin are spe-
cific inhibitors of prokaryotic ribosomes and are effective
agents for the treatment of bacterial infections. Chloram-
phenicol binds to the 50S subunit and inhibits peptidyl
transferase activity, whereas streptomycin binds to the
30S subunit and inhibits chain initiation. Antibiotics such
as puromycin inhibit protein synthesis in both prokaryotes
and eukaryotes. Puromycin mimics amino-acyl tRNA and
enters the A-site of the ribosome where the peptide bond
is formed to release peptidyl-puromycin, which causes
premature termination of protein synthesis. Other eukary-
otic translation inhibitors include cyclohexamide and
diphtheria toxin; they inhibit the elongation step in pro-
tein synthesis.
IMMUNOGLOBULIN
Because a number of different antibodies have been
used for imaging and therapy, we feel it is necessary to at
least introduce the general structure of antibodies in this
article. The major function of immunoglobulin is to bind
to foreign antigen and to inactivate or remove the micro-
organisms or toxins from the body. Antibody production
occurs in B-cells, and our body can generate approxi-
mately 106 different antibodies. Antibodies are Y-shaped
glycoproteins in their monomer form, composed of 4
chains, 2 identical heavy chains (H) and 2 identical light
chains (L). The 4 chains are covalently linked by disul-
phide bonds and each chain is made of 2 principal re-
gions, an N-terminal variable region (V) and a C-terminal
constant (C) region (Fig. 8). The antibody can be cleaved
by a protease called papain to split it into 3 fragments: 2
Fab fragments and 1 Fc fragment. The Fab fragments
contain the variable region and retain the antigen-binding
function, whereas the Fc fragment has the constant region
of the antibody and retains such functions as complement
binding. There are 5 classes of immunoglobulin mole-
cules (IgG, IgM, IgA, IgE and IgD) and they are named
according to the heavy chain they possess (␥, ␮ ␣, ⑀, and
␦, respectively). In addition to the 5 classes of heavy
chains, antibodies can have 2 types of light chains, either
kappa (␬) or lambda (␭) but not both in the same mole-
cule. Each of these chains consists of a single polypeptide
 Reprinted from Academic Radiology, Vol 11, No 4, April 2004
chain of 214 amino acids that can be partitioned into 2
domains, the first consisting of 110 variable amino acids
and called the variable region (V) and the second con-
taining 110 or so amino acids that are species-specific and
which is called the constant (C) region. The variable re-
gion of heavy chains is also 110 amino acids in length,
but the constant region is approximately 330 – 440 amino
acids in length depending on the antibody class. The vari-
able regions in both the light and heavy chains are re-
stricted to 3 small 5–10-amino-acid regions known as the
hyper variable regions or complementarity-determining
regions (CDR). The amino terminal ends of both light
and heavy chains form the antigen-binding site, and the
variability of their amino acid sequences provides the
structural basis for the diversity of antigen-binding sites.
The antibody diversity is generated by DNA recombina-
tion during the development of the B-cell, from 3 pools
of gene segments encoding ␬ ␭ light chains and the heavy
chains. The variable regions of these chains are brought
together by site-specific recombination to generate the
wide repertoire of antibodies. It was in 1976 that Georges
Kohler and Cesar Milstein of the Medical Research Coun-
cil, Cambridge developed the hybridoma technology
where they were able to grow cell lines in vitro that se-
creted large amounts of specific homogenous (monoclo-
nal) antibody that could be easily purified. Recently there
has been an explosion of new biological drugs in clinical
development that are based on recombinant antibodies,
PROTEINS: STRUCTURE AND FUNCTION
Figure 8. Schematic representation of the
immunoglobulin molecule.
and several have recently been approved by the FDA.
Currently a number of different strategies exist for mak-
ing whole IgG therapeutic antibodies. They can be made
from mouse or rat and human chimeric antibodies, or hu-
manized antibodies, which are less immunogenic because
only the complementarity-determing region is of rodent
origin and the rest is human. Fully human antibodies can
also be made in bacteriophage using a technique called
phage display or in a mouse that has had the entire seg-
ment of human immunoglobulin genes cloned into them.
The first therapeutic antibodies marketed in the United
States for non-Hodgkin’s lymphoma and breast cancer
were Rituxan and Herceptin, respectively. Herceptin is the
first humanized antibody approved for the treatment of
HER2-positive metastatic breast cancer; the antibody
blocks the function of HER2 protein. Avastin is another
recombinant humanized antibody that inhibits vascular
endothelial growth factor (VEGF), a protein that plays a
critical role in tumor angiogenesis (the formation of new
blood vessels to the tumor) and maintenance of estab-
lished tumor blood vessels.
PROTEIN DEGRADATION
The cell has evolved mechanisms to carefully control
and regulate the level of proteins. Enzymes, cell cycle
proteins such as cyclins, damaged or misfolded proteins,
and other elements are all degraded by complex mecha-
S39
 PANDIT AND LI
nisms that exist in the cell, and the rate of degradation
determines their concentration in the cell. The protein
degradation process requires ATP, is energy-dependent,
and occurs mostly in the lysosomes or cytosol. Addition-
ally, the endoplasmic reticulum is the site of degradation
of misfolded secretory proteins and excess subunits of
cell surface receptors. Most non-selective protein degrada-
tion takes place in the lysosomes, where changes in the
supply of nutrients and growth factors can influence the
rates of protein breakdown. Lysosomes are small or-
ganelles that contain a number of hydrolytic enzymes and
approximately 40 different acid hydrolases, including pro-
teases, nucleases, glycosidases, lipases, and others. The
pH inside the lysosome is acidic (pH ⫽ 5) because the
enzymes require an acid environment for optimal activity.
Short-lived regulatory proteins such as cyclins are re-
quired in this particular phase of the cell cycle and are
then destroyed in the cytosol by local proteolytic mecha-
nisms. All short-lived proteins are thought to contain rec-
ognition signals, presumably amino acid sequences or
conformational motives that differ from the normal pro-
teins and hence tag them for early degradation. These
proteins are recognized by the ubiquitin-dependent pro-
teolytic systems, which selectively label proteins that are
targeted for degradation with ubiquitin molecules (11).
Ubiquitin is a 76-amino-acid protein that binds covalently
to available lysine residues on target proteins. In subse-
quent steps, the ubiquitin-conjugating enzyme adds a se-
ries of additional ubiquitin molecules to the attached
ubiquitin to form a multiubiquitin complex, which is rec-
ognized by the large multisubunit 26S proteasome com-
plex that cuts the target protein into a series of small frag-
ments.
SUMMARY
This article along with the first 2 in this series (4,12)
completes the discussion on the key molecules and process
inside the cell namely, DNA, RNA, and proteins. These 3
articles provide a very basic foundation for understanding
molecular biology concepts and summarize some of the
work of numerous scientists over the past century. We un-
derstand these processes far better now than we did in the
past, but clearly this knowledge is by no means complete
and a number of basic scientists are working hard to eluci-
date and understand the fundamental mechanisms that oper-
ate within a cell. Genes and gene products work with each
other in complex, interconnected pathways, and in perfect
harmony to make a functional cell, tissue, and an organism
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Reprinted from Academic Radiology, Vol 11, No 4, April 2004
as a whole. There is a lot of cross-talk that happens between
different proteins that interact with various other proteins,
DNA, and RNA to establish pathways, networks, and molec-
ular systems as a team working to perfection. The past 15
years have seen the rapid development of systems biology
approaches. We live in an era that emphasizes multi-disci-
plinary, cross-functional teams to perform science rather than
individual researchers working on the bench on a very spe-
cific problem. Global approaches have become more com-
mon and the amount of data generated must be managed by
trained bioinformatics personnel and large computers. In our
subsequent articles, we will discuss these global approaches
and the areas of genomics, functional genomics, and pro-
teomics that have revolutionized the way we perform sci-
ence.
GLOSSARY
Aminoacyl-tRNA synthetase: An enzyme that is re-
sponsible for covalently linking amino acids to the 2⬘ or
3⬘ hydroxyl (-OH) group on the tRNA molecule.
Chaperones: Proteins such as heat shock protein 60
(hsp60) and hsp70 are called chaperone proteins because
they work with a small set of other proteins and help
other proteins to fold correctly.
Conformation: The spatial arrangement of substituent
groups that are free to assume many different positions
without breaking bonds, because of rotation around the
single bonds in the molecule.
Peptidyl transferase: The enzyme that catalyzes the
synthesis of the peptide bond between the polypeptide
attached to the P-site and the amino-acyl tRNA present in
the A-site of the ribosome.
Polyprotein: A single translation product that is subse-
quently processed by proteases to generate 2 or more sep-
arate proteins. For example, ubiquitin is synthesized as a
polyubiquitin and then processed into single molecules.
Polysomes: Elements formed when multiple ribosomes
bind to the same mRNA molecule and begin translating
and moving along the mRNA.
Shine-Dalgarno sequence: A 4 –7 base-long polypurine
stretch of sequence (5⬘-AGGAGG-3⬘) that precedes the
AUG initiation codon in prokaryotic mRNAs. This sequence
helps the prokaryotic ribosome to align itself and initiate
translation beginning at the first AUG codon after it.
Signal recognition particle (SRP): A particle made of 6
polypeptides and a small RNA molecule that recognizes
the signal peptide sequence and guides the polypeptide to
the endoplasmic reticulum.
 Reprinted from Academic Radiology, Vol 11, No 4, April 2004 PROTEINS: STRUCTURE AND FUNCTION

REFERENCES ADDITIONAL REFERENCE MATERIAL

1. National Center for Biotechnology Information. Available at: http://
www. ncbi.nlm.nih.gov/entrez/query.fcgi?db⫽Protein. Accessed • Alberts B, Bray D, Lewis J, Raff M, Roberts K,
December 30, 2003.
2. Database of protein families and domains. Available at: http://us.expasy. Watson JD. Molecular Biology of the Cell.
org/prosite/. Accessed December 30, 2003. 3rd ed. New York: Garland Publishing;
3. National Human Genome Research Institute (NHGRI) project on Ency-
1994.
clopedia of DNA Elements. Available at: http://www.genome.gov/
Pages/Research/ENCODE. Accessed December 30, 2003. • Griffiths AJF, Miller JH, Suzuki DT, Lewontin RC,
4. Pandit SD, Bednarski M, King Li. A primer on molecular biology for Gelbart WM. Introduction to Genetic Analysis. 7th ed.
imagers: I. DNA, how does it work? Acad Radiol 2003; 10:1215–1223.
5. Basharov MA. Protein folding. J Cell Mol Med 2003; 7:223–37.
New York: W H Freeman & Co; c1999.
6. Swanton E, Bulleid NJ. Protein folding and translocation across the • Griffiths AJF, Gelbart WM, Miller JH, Lewontin RC.
endoplasmic reticulum membrane. Mol Membr Biol 2003; 20:99 –104. Modern Genetic Analysis. New York: W H Freeman &
7. Young JC, Barral JM, Ulrich Hartl F. More than folding: localized func-
tions of cytosolic chaperones. Trends Biochem Sci 2003; 28:541–7.
Co; c1999.
8. Aguzzi A, Haass C. Games played by rogue proteins in prion disorders • Lodish H, Berk A, Zipursky SL, Matsudaira P, Balti-
and Alzheimer’s disease. Science 2003; 302:814 – 8. more D, Darnell JE. Molecular Cell Biology. 4th ed.
9. National Center for Biotechnology Information. Available at: http://
www.ncbi.nlm.nih.gov/Structure/. Accessed December 30, 2003. New York: W H Freeman & Co; c1999.
10. Protein Data Bank for 3-D biological macromolecular structure data. • Lewin B. Genes VII. 2000. Oxford: Oxford University
Available at: http://www.rcsb.org/pdb/. Accessed December 30, 2003.
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11. Ben-Neriah Yinon. Regulatory functions of ubiquitination in the im-
mune system. Nat Immunol 2002; 3:20 – 6. • Cooper GM. The Cell: A Molecular Approach. 2nd ed.
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tion and gene expression analysis. Acad Radiol 2004; 11:333–344.

S41

A Primer on Molecular Biology for Imagers:
IV. Concepts and Basic Methods in Molecular Biology1
In this review article series so far, we have covered the
three fundamental molecules: DNA, RNA, and proteins,
and some of the basic processes inside the cell that these
molecules are part and parcel of (1,2,3). The challenge
for us in terms of writing a review article on “methods in
molecular biology” is how to make it interesting and not
a drab rendition of procedures, many examples of which
are already available. Keeping this in mind, we have cov-
ered some of the very basic procedures that a radiologist,
molecular imager, chemist, or physicist as a part of a
multi-disciplinary team in molecular imaging would be
required to know. We have attempted to emphasize the
principles behind the molecular biology protocols rather
than the procedures themselves.
DNA METHODS
DNA Isolation
There are a number of methods that are available to
isolate genomic and plasmid DNA. The genomic DNA
isolation methods must be gentle to avoid DNA denatur-
ation and shearing, whereas the most common method to
isolate plasmid DNA that is circular and more resistant to
denaturation is the alkaline lysis method or one of its
modifications. In either case, the first step is to have suffi-
cient cells or tissue from which the genomic DNA must
be isolated. In the subsequent steps the cells or tissue are
lysed using a cell-lysing buffer, which may contain pro-
teases such as proteinase K, and detergent such as sodium
Reprinted with permission from Acad Radiol 2004; 11:686 – 697
1 From the Molecular Imaging Laboratory, Department of Diagnostic Radi-
ology, Clinical Center, 10/1N306, National Institute of Health, 9000 Rock-
ville Pike, Bethesda, MD 20892 (S.D.P., K.C.P.L.). Received ; accepted.
Address correspondence to S.D.P. e-mail: spandit@mail.cc.nih.gov
© AUR, 2004
doi:10.1016/j.acra.2004.10.005
S42
Chapter IV
Sunil D. Pandit, PhD, King C.P. Li, MD, MBA
do-decyl sulphate (SDS). Cell lysis releases all the con-
tents of the nucleus and cytoplasm including DNA, RNA,
and proteins. The RNA is degraded by treatment with
RNase A, and the proteins are degraded by proteinase K
treatment and removed following phenol:chloroform treat-
ment. The chromosomal DNA is precipitated using etha-
nol in the presence of salts such as sodium acetate
(NaOAC). The chromosomal DNA precipitates out in a
string form, and larger isolation quanities can actually be
wound around a glass rod. The DNA is dried and re-sus-
pended, preferably in a Tris-EDTA buffer (TE). The
EDTA chelates Mg⫹2 ions, which are required for the
activity of DNase1 enzyme, which nicks DNA and hence
prevents DNA degradation. The protocols for plasmid
isolation are based on the alkaline lysis procedure. Plas-
mids, as we will discuss later, are propagated in bacteria.
Large amounts of bacteria can be grown in the laboratory
and the plasmid DNA isolated from the bacterial cells.
Bacteria are lysed with SDS and sodium hydroxide,
which denatures the double-stranded (ds) chromosomal
DNA. Treatment with potassium acetate and acetic acid
causes an insoluble precipitate of detergent, phospholip-
ids, and proteins and neutralizes the sodium hydroxide. At
neutral pH, the long strands of chromosomal DNA re-
nature only partially and become trapped in the precipi-
tate, which is removed by centrifugation. The plasmid
DNA re-natures completely and remains in solution. Etha-
nol or isopropanol is added to the supernatant to precipi-
tate the plasmid DNA out of the solution. Following cen-
trifugation, the DNA pellet is washed with 70% ethanol
and dissolved in water or buffer such as TE.
We routinely use kits that use column purification
methods (Qiagen, Germantown, MD) to perform mini and
maxi preparations of plasmid DNA. Following alkaline
lysis, the plasmid DNA is bound to an anion-exchange
resin under appropriate low salt and pH conditions.
Proteins, RNA, and other impurities are removed by
 Reprinted from Academic Radiology, Vol 11, No 6, June 2004 CONCEPTS AND BASIC METHODS IN MOLECULAR BIOLOGY
medium-salt wash. Plasmid DNA is isolated using a high-
salt buffer and then precipitated with isopropanol. Endo-
toxin or lipopolysaccharides (LPS) are components of the
outer layer of bacterial membrane that are released into
the lysate during DNA preparations and must be avoided.
For animal studies and in vivo injection, an endotoxin-
free DNA preparation is required because endotoxins
cause fever, endotoxic shock syndrome, and activation of
complement cascade in animals and humans.
RESTRICTION ENZYMES AND GEL
ELECTROPHORESIS
Restriction Endonucleases
In 1970, Hamilton Smith and Kent Wilson at John
Hopkins University isolated the first type II restriction
endonuclease. Type II restriction enzymes are extremely
useful for the molecular biologist because they cut DNA
at a precise position within its recognition sequence and
have no modifying activity. More than 200 restriction
endonucleases covering at least 100 different recognition
sites are commercially available. The name given to each
enzyme reflects its origin. For example, the endonuclease
BamH1 was isolated from the bacteria Bacillus amyloliq-
uefaciens. The number 1 indicates that it was the first
endonuclease to be identified from these bacteria.
Restriction endonucleases usually recognize palin-
dromic 4- to 8-nucleotide sequences. Most of these en-
zymes function as dimers. Some enzymes cut both strands
symmetrically in the middle of the target sequence, gener-
ating “blunt ends,” while others cut both strands off-cen-
ter, generating staggered ends. For example BamH1 rec-
ognizes the sequence:
5⬘-GGATCC-3⬘
3⬘-CCTAGG-5⬘
BamH1 cuts after the first G in both strands, generating
fragments with the following ends:
5⬘-G GATCC-3⬘
3⬘-CCTAG G-5⬘
The single-strand– overhanging ends are complementary
to the DNA on the end cut by the same enzyme. These
“sticky” or cohesive ends are extremely useful for “cut-
ting and pasting” DNA from different origins and there-
fore creating recombinant DNA.
Agarose Gel Electrophoresis
This is a simple and rapid method to separate DNA
fragments according to their size. The DNA sample,
along with gel loading dye (a mixture of Bromophenol
blue—fast migrating dye, Xylene Cyanol—slow migrating
dye, and glycerol), is loaded into a well in a gel immersed
in buffer and is electrophoresed. The DNA molecules, being
negatively charged, migrate from the negative to the positive
terminal and most of the DNA migrates between the slow-
and fast-migrating dyes. The porous gel matrix acts as a
sieve through which larger molecules move with more diffi-
culty than smaller ones. The distance run by a DNA frag-
ment is inversely proportional to its molecular weight and
therefore to its length or number of nucleotides. The gel is
commonly stained with a DNA interchelating dye such as
ethidium bromide, which interchelates with the DNA. The
DNA is visualized using UV light. The ethidium bromide
absorbs UV radiation at 302 nm and 366 nm and emits at
590 nm in the red– orange region of the visible spectrum.
The lab on the chip (Agilent Technologies, Palo Alto, CA)
is replacing the traditional agarose gel electrophoresis for
DNA and RNA (Figure 1).
MOLECULAR CLONING
In 1972, Stanley Cohen, Herb Boyer, and Jeremy Berg
developed recombinant DNA technology, which allowed
for the DNA from one organism (eg, humans) to be
cloned into a carrier DNA molecule called a vector and
expressed in the new host. The new DNA molecule, a
“recombinant” of the original DNA with the vector DNA,
could now replicate and express the gene in the new host
because of the universality of genetic code. This tech-
nique called “molecular cloning” has revolutionized the
molecular analysis of the structure and function of eu-
karyotic genes and genomes.
Vectors
A vector is a DNA molecule used to carry a foreign
DNA fragment into a host organism, which can be a bac-
terium or a eukaryotic host. There are a number of differ-
ent kinds of vectors used by molecular biologists, but we
will focus our attention on plasmid vectors.
Plasmids
Plasmids are the simplest kind of circular DNA vectors
that can replicate independently from the chromosome.
There is a vast selection of commercially available plas-
mids such as those useful for DNA sequencing, protein
expression in bacteria or in mammalian cells (Figure 2),
and those used for reporter gene expression such as green
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 PANDIT AND LI Reprinted from Academic Radiology, Vol 11, No 6, June 2004

Figure 1. Lab on a chip. Agi-

lent Technologies has developed
these DNA and RNA chips that
are shown along with Bioana-
lyzer machine. The chips use
microfluidics technology and are
an easy and rapid way to check
the DNA restriction digestion or
RNA quality. Representative ex-
amples of DNA and RNA analy-
sis are shown. DNA restriction
fragment analysis computes the
size of the fragments by com-
paring them to the DNA ladder
and also determines their con-
centration. For total RNA analy-
sis, the ratio of readings at 260
nm and 280 nm is determined. It
should be in the 1.9 –2.1 range;
the ribosomal RNA bands 28S
and 18S should appear sharp
and intact. If they appear
smeared, there is likely degrada-
tion of RNA during preparation.

Figure 2. Prokaryotic and eukaryotic plasmid vectors.

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 Reprinted from Academic Radiology, Vol 11, No 6, June 2004 CONCEPTS AND BASIC METHODS IN MOLECULAR BIOLOGY
fluorescent protein (GFP), luciferase gene (used by optical
imagers) and others.
Each plasmid contains an origin of replication, which
allows it to replicate independently of the host DNA, and
a selectable marker that encodes for antibiotic resistance
and allows cells containing the plasmid to be selected.
Examples of antibiotics used for bacterial cloning and
selection of recombinants are ampicillin and kanamycin,
those for eukaryotic vectors include neomycin, and those
suitable for prokaryotic and eukaryotic hosts include anti-
biotics such as zeocin. Eukaryotic vectors also contain
bacterial-selectable markers (shuttle vectors) so that they
can be propogated in both prokaryotic and eukaryotic
hosts. Most plasmids contain cloning sites, also called
polylinkers, which are a series of unique recognition se-
quences for different restriction endonucleases. Addition-
ally, the plasmid may contain a promoter that precedes
the polylinker site and drives the expression of the insert
cloned into the polylinker site. Typical examples of pro-
karyotic and eukaryotic plasmids are shown in Figure 2.
In order to clone a DNA fragment it must be inserted
into the appropriate vector. The choice of vectors and re-
striction sites must be planned ahead. In a simple cloning
strategy (Figure 3), the vector DNA (eg, plasmid) is digested
with a chosen restriction enzyme that cuts once at the
polylinker. The DNA fragment to be inserted (insert) is cut
with the same enzyme. The restriction-digested products are
usually electrophoresed on a preparative agarose gel along
with molecular size markers, and the vector and insert bands
are gel purified. The sticky ends of the linearized plasmid
can form hydrogen bonds with the complementary nucleo-
tides present in the overhang of the DNA fragment to be
inserted. The vector and insert DNA fragments are ligated
together in the presence of DNA ligase, which forms phos-
pho-diester bonds between adjacent nucleotides in the appro-
priate buffer and temperature conditions The purified vector
and insert DNA are mixed together in a molar ratio of ap-
proximately 10:1 (insert:vector) and ligated at a lowered
temperature (15°C) to provide sufficient excess of insert to
be inserted into the two ends of the vector. This allows the
intermolecular ligation of the vector and insert to be more
kinetically favored over the intramolecular ligation of the
sticky ends of the vector.
Some of the modern topoisomerase vectors (Invitrogen,
Carlsbad, CA) take advantage of the single nucleotide
dA-tail that is generated at the ends of PCR products.
These PCR products can be efficiently and quickly ligated
to the dT-overhangs on the Topo-vectors, facilitated by
the presence of DNA topoisomerase.
Other Vectors
Typical plasmid vectors range in size from 3–5 kb and
can accommodate inserts up to 15 kb in length. Several
other vectors are available for cloning fragments of DNA
of different sizes depending on the type of host and appli-
cation. They include ␭ vectors that can accommodate for-
eign DNA 5 to 14 kb in length. The recombinant DNA is
“packaged” into viral particles and used to infect Esche-
richia coli. The cloning of the entire human genome in
overlapping sets of fragments required the development of
specialized vectors. Cosmids are one such vector that is
useful for producing large-insert genomic libraries. They
replicate similar to a low-copy plasmid and accept DNA
fragments from sizes of 28 to 45 kb in length. Other vec-
tors, such as bacterial artificial chromosomes (BACs), are
capable of carrying large fragments up to 350 kb of eu-
karyotic DNA in bacteria. Yeast artificial chromosomes
(YACs) are propogated in the Baker’s yeast Saccharomy-
ces cerevisiae and Mega-YACs, as the name suggests,
have insert sizes up to 1 megabase of human or mouse
genomic or other eukaryotic DNA.
THE HOST CELL
Transformation of Bacteria
The recombinant DNA molecules that are produced in
vitro following ligation are now ready to be introduced
into a living cell such as E. coli by a process called trans-
formation. The transformation process uses bacteria that
are made competent to modify the cellular membrane by
treatment with chemicals such as DMSO. The ligation
mixture and the competent cells are incubated together on
ice, and ligated DNA molecules (now circular) are taken
up by the competent bacteria when subjected to a mild
heat shock at 42°C for 1–2 min. Other methods of trans-
formation include passing high voltage current through
the ligation mixture and competent cells, which intro-
duces the DNA into competent cells. The cells are
grown in a rich media such as Luria-Bertani (LB) me-
dia and plated on a LB agar plate with the appropriate
antibiotic for selection of the recombinant clones. Only
one type of molecule of the recombinant plasmid in the
ligation mix transforms a single bacterial cell; in other
words multiple different types of molecules cannot rep-
licate inside a single bacteria. This provides efficient
selection of individual clones, and after 16 –24 hours
individual colonies, each representing a single mole-
cule of the recombinant clone, are obtained on an anti-
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 PANDIT AND LI Reprinted from Academic Radiology, Vol 11, No 6, June 2004

Figure 3. A schematic diagram of cloning and

transformation strategy. The vector and insert DNA
are cut with appropriate restriction enzymes and
ligated. This results not only in the vector and in-
sert ligating together but (if only a single restriction
site is used) will also result in the vector ligating
together. Such a mixture when transformed into
bacteria will result in mixture of untransformed,
vector-transformed, and recombinant-transformed
bacteria. Only the latter two will grow on an antibi-
otic selection plate and form individual colonies.
The recombinant is screened by DNA purification
and checking for the presence of insert.

Figure 4. Plasmids for optical imaging. Optical reporter genes such as enhanced green fluorescent protein
(EGFP) and Discosoma Red protein containing plasmids are shown. Bioluminescent reporter gene plasmids from
Firefly and Renilla are also shown. Expression of the reporter genes is driven by strong CMV or SV40 promoters.
These vectors have convenient multiple cloning sites (MCS) for cloning other genes and expressing reporter-
fusion proteins.

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 Reprinted from Academic Radiology, Vol 11, No 6, June 2004 CONCEPTS AND BASIC METHODS IN MOLECULAR BIOLOGY
biotic selection plate. Individual colonies are picked
and further propagated in liquid LB culture medium
with antibiotic, the DNA isolated and the clones
checked for the presence of the inserted gene.
Transfection of Eukaryotic Cells
There are a variety of methods available for the deliv-
ery of genes into eukaryotic cells. They fall into three
categories: biochemical methods, physical methods, or
transduction mediated by viruses. Physical methods in-
clude electroporation, where a pulsed electric field is ap-
plied to create transient pores in the plasma membrane
and to introduce DNA into cells. Other physical methods
include direct injection of the DNA into the cell. Bio-
chemical agents of transfection include calcium phosphate
and DEAE dextran, which has been used for a long time.
Recently, there are several cationic liposome– based tran-
fection agents such as Lipofectamine2000 (Invitrogen),
superfect (Qiagen), FuGEN6 (Roche Diagnostics, Basel,
Switzerland), poly-L-Lysine, and others that have been
successfully used to deliver genes into a wide variety of
cell lines. Lipid-based transfection agents are mixed with
DNA and the lipid forms a complex with the DNA when
the DNA is coated with the lipid, which is taken by the
plasma membrane of the cell or is taken into the cell us-
ing non-receptor mediated endocytosis. The efficiency of
transfection varies with the cell line; DNA quality; and
the nature of transfection, whether stable or transient. In
transient transfection, recombinant DNA is introduced
into a cell line in order to obtain a temporary high-level
expression of the gene. The transfected DNA does not
integrate into the host chromosomal DNA. In stable trans-
fection, the transfected DNA integrates into the host chro-
mosomal DNA and expresses the target protein. Stable
transfectants are selected in a background of untransfected
cells using a selectable drug resistance marker.
COMMONLY USED REPORTER–PROBE
SYSTEMS IN MOLECULAR IMAGING
The two most common imaging modalities that use
reporter probe systems, at least in small animals, include
optical and positron emission tomography (PET) imaging.
The reporter genes are usually linked to the therapeutic
genes to monitor the expression of therapeutic genes. The
two techniques in optical imaging that have become pop-
ular are fluorescence and bioluminescence imaging. In
fluorescence imaging, energy from an external source of
light is absorbed and immediately re-emitted at a longer,
lower-energy wavelength. The green fluorescent protein
(GFP) from Aequorea victoria has been mutated exten-
sively to generate variants with improved properties and
altered colors. There are a number of different colored
proteins available namely, green (GFP), blue (BFP), cyan
(CFP), and yellow fluorescent proteins (YFP). Recently,
long-wavelength red fluorescent proteins have been identi-
fied from the Discosoma genus, such as DsRed2, and
mRFP1. Vectors expressing these genes are commercially
available and examples of these vectors are shown in Fig-
ure 4. Fluorescent protein– based indicators have an ad-
vantage in that they can be introduced into a wide variety
of tissues and intact organisms and have allowed the in-
vestigation of more complex processes inside the cell.
Bioluminescence is a subset of chemi-luminescence, in
which the light-producing chemical reaction occurs inside
an organism. Bioluminescence imaging is used to detect
the photons emitted from cells that have been genetically
engineered to express luciferases. Luciferases comprise a
family of photoproteins isolated from a variety of species
that modify their substrates causing the release of pho-
tons. Commonly used luciferase genes have been identi-
fied from the firefly (Photinus pyralis) and Renilla (Re-
nilla reniformis), a sea pansy. The firefly luciferase pro-
tein uses luciferin as the substrate whereas the Renilla
luciferase uses Coelenterazine. Luciferase can be trans-
genically expressed in mammalian cells, and when ex-
posed to its substrate, releases photons that can be de-
tected and counted using a camera.
PET is another method that allows an indirect mea-
surement of the expression of linked therapeutic genes of
interest. The therapeutic gene and the reporter gene can
be placed under two different promoters in the same vec-
tor. Alternatively, a bicistronic vector with an internal
ribosomal entry site (IRES) can be used. This usually
results in a lower expression of the downstream gene.
Thirdly, the reporter and the therapeutic genes can be
fused together, but one must be careful because this can
result in poor expression and activity of the two fused
genes. The most commonly used PET reporter gene sys-
tem is the HSV1–tk gene (Herpes Simplex Virus–thymi-
dine kinase gene). Cells overexpressing the HSV1–tk gene
phosphorylate the tracer acycloguanosines derivatives
[such as ganciclovir and penciclovir and derivatives of
thymidine such as FIAU (2⬘-fluoro-2⬘-deoxy-1-␤-D-ar-
abinofuranosyl-5-iodo-uracil)], which gets trapped inside
the cell and can be imaged.
S47
 PANDIT AND LI
The Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a very useful
in vitro method to exponentially amplify small amounts
of DNA fragment or gene. This method, for which a No-
bel prize was awarded, was invented by Kary Mullis in
1985. In PCR, DNA is replicated in a test tube, replacing
the steps that normally occur within a cell. All that is
needed is a small amount of DNA to be amplified (as
little as a few molecules) and sequence information of
approximately 15–20 nucleotides at the ends of the DNA
fragment, which is used to design primer sequences with
similar melting temperatures (Tm) and GC content
(⬃50%). The PCR technology was feasible because of
the discovery of thermostable DNA polymerase from the
organism Thermus aquaticus. The two primers, a DNA
sample containing the target sequence, Taq DNA poly-
merase, the four deoxyribonucleoside triphosphates
(dNTPs), and a magnesium buffer of appropriate concen-
tration are mixed together and thermal-cycled in a pro-
grammable machine called a thermal cycler (Figure 5). In
the first step, called denaturation, the template DNA is
denatured and the two strands of the double-stranded
DNA are separated at high temperature (94°). In the next
step, called annealing, the temperature is lowered to allow
the two primers to anneal to complementary strands on
the target. The annealing temperature is usually between
50°C and 60°C and varies for different primers depending
on their Tm. In the third extension step, the Taq DNA
polymerase extends the primers (usually at 72°C) at the
3⬘-OH end, incorporating nucleotides using the target
DNA as a template, and creates a DNA strand comple-
mentary in sequence to the strand the primers annealed.
The cycles of denaturation, annealing, and extension are
repeated many times, usually in 30 – 40 cycles, which
leads to the exponential amplification of the DNA product
flanked by the primers. The primers anneal to comple-
mentary DNA sequences at the 5⬘ end of each strand of
the DNA fragment and the products obtained have the
sequence of the template bracketed by the primers. For
example, a single DNA molecule amplified through 30
cycles of replication would theoretically yield 229 progeny
molecules. The PCR products can be checked rapidly on
an agarose gel, and the band of expected size with very
little background and no bands in the negative control (no
template DNA) is indicative of a successful PCR. Normal
PCR can amplify products up to a few kilobases, whereas
there are long-range PCR reagents available (Elongase
Taq) that can amplify products up to 40 kb with high fi-
delity and a low error rate. All the PCR reagents are
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Reprinted from Academic Radiology, Vol 11, No 6, June 2004
available in kit format, including PCR master mixes that
require only the addition of primers and template DNA.
DNA SEQUENCING
Modern sequencing is based on the di-deoxy chain
termination method developed by Fred Sanger in 1977,
for which he won his second Nobel prize (awarded in
1980). The Sanger method uses in the initial step a short
primer that is hybridized to a DNA template in the pres-
ence of the four dNTP’s, and a DNA polymerase is able
to synthesize a new strand of DNA complementary to the
template. Additionally, 2⬘-3⬘-dideoxyribonucleotide
triphosphates (ddNTP’s) are included in the reaction, and
chain elongation by DNA polymerase terminates when a
ddNTP is incorporated because the ddNTP lacks the 3⬘
hydroxyl group necessary to form the phosphodiester
linkage with the next nucleotide to be incorporated. The
present-day automated fluorescent cycle sequencing
method uses fluorophore-labeled ddNTPs, and the reac-
tion is carried out in a thermocycler. The reaction mix
contains a DNA template, a primer, Taq DNA polymer-
ase, the four dNTP’s, and four ddNTP’s, each labeled
with a different fluorescent dye (Figure 6). The primer is
designed so that it is complementary to the beginning of
the fragment to be sequenced, or if the gene is cloned in
a plasmid, the primer anneals to a region upstream and
close to the cloning site of the gene. The cycle sequenc-
ing reaction uses a two-step cycle of denaturation at 94°C
and annealing and extension at 65°C repeated for 25–35
cycles. The DNA polymerase randomly adds dNTP’s or
ddNTP’s that are complementary to the DNA template.
Each time a ddNTP is incorporated, the chain extension
by DNA polymerase terminates and a DNA fragment is
produced that has a discrete size and is fluorescently la-
beled according to its last nucleotide. The sequencing
products differing in length by a single base (terminated
at each base) are separated on a matrix of polyacrylamide
gel and urea (denaturing agent), creating a “ladder” of
DNA fragments, each longer than the previous one by
one nucleotide. The fluorescently labeled products are
detected as the terminated fragments pass a laser aimed at
the bottom of the gel. Each fluorescent terminator emits
at a particular wavelength when excited by the laser, and
the information is collected and interpreted by a com-
puter. Modern sequencers use multiple capillary electro-
phoresis and proprietary polymer material instead of a gel
slab and polyacrylamide, allowing complete automation
of the electrophoresis and data collection process.
 Reprinted from Academic Radiology, Vol 11, No 6, June 2004 CONCEPTS AND BASIC METHODS IN MOLECULAR BIOLOGY

Figure 5. A schematic drawing of polymerase chain reaction (PCR). The target DNA (in blue) and the target region to be amplified
(boxed) are shown. The primers anneal to the complementary sequences and the multiple cycles of denaturation, annealing, and exten-
sion result in the exponential amplification of the target region. The amplified product plateaus, after a certain number of cycles, the level
of which depends on the initial number of target sequences, the quantity of reagents, and the efficiency of extension.

Figure 6. DNA Sequencing. A representative

example of fluorescent dye-deoxy termination
sequencing is shown.

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 PANDIT AND LI
Equipped with robotic sample transfer arms, these ma-
chines (ABI3700; Applied Biosystems, Foster City, CA)
can potentially electrophorese 4 ⫻ 384 samples without
manual intervention. Hundreds of these machines running
continuously were the workhorses behind the complete
sequencing of the human genome and other model organ-
isms.
DETECTION OF DNA
Southern Blotting
This technique, developed by E.M. Southern in 1975,
is based on the ability of the complementary base se-
quences to recognize each other and to form a reversible
chemical bond (Figure 7). The DNA to be analyzed is
digested with one or more restriction endonucleases, and
the fragments along with appropriate size standards are
separated by agarose gel electrophoresis. The DNA frag-
ments are denatured with NaOH and transferred or “blot-
ted” to a membrane such as nitrocellulose or nylon and
cross-linked using UV light or high temperature. The
membrane-bound fragments have the same relative posi-
tions as the fragments separated by size on the gel. The
filter is then hybridized under appropriate salt and temper-
ature conditions with a labeled probe, and the fragments
containing the sequence of interest are detected as labeled
bands. The labeled probe is made out of DNA or RNA
that is complementary to the target DNA to be detected
and is immobilized on the membrane. The probes can be
radioactively labeled probes or non-radioactive chemilu-
minescent probes.
RNA METHODS AND CONCEPTS
RNA Isolation and Quantification
One of the key prerequisites for good quality RNA
isolation, particularly from tissues, is to stabilize RNA
because there is non-specific and specific RNA degrada-
tion following the harvesting of the biological sample.
This can be minimized by rapidly freezing the tissue sam-
ple in liquid nitrogen or in RNA stabilization reagents
such as RNAlater (allowing the transport and storage of
the sample at ambient temperatures). Biological samples
are first lysed and homogenized in the presence of a gua-
nidine isothiocyanate (GITC)– containing buffer, which
inactivates RNases to ensure isolation of intact RNA.
DNA and proteins are removed by centrifugation and the
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Reprinted from Academic Radiology, Vol 11, No 6, June 2004
remaining DNA can be removed by treating with DNase
I. The total RNA is bound to the column (Qiagen),
eluted, resuspended in RNase-free water [Diethyl pyro-
carbonate (DEPC)–treated] and quantified. mRNA con-
stitutes only 1%–2% of the total cellular RNA, con-
tains a poly (A) tail, and can be purified from total
RNA using a oligo-dT column that binds to the poly
(A) tail. The unbound cellular RNA is washed away
and the mRNA eluted from the column. Purified RNA
is very sensitive to degradation because of its single-
stranded nature and also because of the stability of
RNaseA that is commonly used in DNA preparations
and can easily contaminate the RNA preparations.
Hence, most labs have a separate area for RNA analy-
sis, and all glassware and pipettes must be RNase free.
Glassware and other material used for RNA work are
treated with diethyl-pyrocarbonate (DEPC), which in-
hibits RNases. All RNA preparations are stored at
⫺80°C and preferably in small aliquots to prevent mul-
tiple freeze–thaws and degradation.
DETECTION OF RNA
Northern Blotting
This technique, a variation of the Southern blotting, is
used for detection of RNA instead of DNA and gives an
estimate of the level of gene expression and the size of
the RNA. Total cellular RNA or purified mRNA is size-
fractionated through denaturing agarose gel electrophore-
sis. The presence of denaturing agents such as formalde-
hyde and others prevents the formation of secondary
RNA structures during electrophoresis. The RNA is then
blotted onto a filter and detected by hybridization with a
labeled probe.
Reverse Transcription-Polymerase Chain
Reaction
Reverse transcription-polymerase chain reaction (RT-
PCR) combines the first steps of cDNA synthesis with
reverse transcription to amplify cDNA copies of RNA. In
the initial step, an oligo-dT primer, random hexamer
primer (pdN6), or a gene-specific primer is hybridized to
the target gene mRNA or all mRNA (if using oligo-dT or
random hexamers) and is extended by an RNA-dependent
DNA polymerase (reverse transcriptase). The generated
cDNA is amplified using specific primers. This method
can be used to determine the strength of gene expression
when the RNA available is limited or expressed at very
low levels.
 Reprinted from Academic Radiology, Vol 11, No 6, June 2004 CONCEPTS AND BASIC METHODS IN MOLECULAR BIOLOGY

Figure 7. Southern Blotting.

The target DNA is restriction di-
gested and electrophoresed on
an agarose gel. The DNA is de-
natured using alkali and trans-
ferred to a membrane such as
nitrocellulose using passive blot-
ting or vacuum. The DNA is then
cross-linked to the membrane
using heat or UV light, and the
membrane is hybridized to a la-
beled probe. The excess probe
is washed and the signal from
the hybridized probe is detected
using autoradiography.

Figure 8. TaqMan analysis.

The fluorophore– quencher pair
containing primer (TaqMan
probe) must be designed in such
a way that it is highly specific to
the target gene. Initially, the two
primers and the TaqMan probe
anneal to the target sequence.
As the primer is extended, the
exonuclease activity of the poly-
merase digests the TaqMan
probe, the fluorophore, and the
quencher pair fall apart and are
no longer present at a fixed dis-
tance from each other (so that
the quencher can quench the
fluorescence). In subsequent
rounds of each, the amount of
free fluorophore increases expo-
nentially, which is detected and
measured quantitatively by the
detector.

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Figure 9. A schematic repre-

sentation of cDNA synthesis.
Oligo-dT (shown) or random
primer is used to anneal to the
mRNA. Reverse transcriptase
extends and synthesizes the first
strand of the cDNA. Treatment
with RNase H and DNA polymer-
ase 1 nicks the RNA in the DNA:
RNA hybrid. This creates primers
with 3⬘-OH groups that are ex-
tended by T4 DNA polymerase
to complete the second strand
cDNA synthesis. Linkers with
rare restriction endonuclease
sites are blunt end ligated to the
cDNA ends and the ds cDNA
product is cloned into a vector
of choice (not shown).

Real-time PCR
Real-time PCR is used to measure the abundance of
particular DNA or RNA sequences, to quantify gene ex-
pression, and to confirm differential expression of
genes detected by microarray technology. In real-time
PCR, commercially available fluorescence-detecting
thermocyclers (ABI5700, ABI7900 [Applied Biosys-
tems] lightcycler, etc.) are used to amplify specific nu-
cleic acid sequences and the product concentration is
measured simultaneously. DNA interchelating dyes
such as SYBR green are used to estimate at any time
point the amount of fluorescence and hence the yield of
amplified DNA. The TaqMan method (Figure 8) uses a
fluorophore and quencher containing specific oligonu-
cleotide to anneal to an internal sequence in the ampli-
fied DNA fragment. As the amount of target DNA in-
creases, progressively greater quantities of the oligonu-
cleotide probe sequence anneal to the target. During
the extension phase of PCR, the 5⬘3 3⬘ activity of the
Taq polymerase cleaves the fluorophore from the
probe, and the fluorophore is now separated from the
quencher. The intensity of the fluorescence of the sam-
ple is directly proportional to the amount of target
DNA synthesized. The chief advantages of real-time
PCR are its high sensitivity and the ability to process
many samples simultaneously and rapidly.
CDNA SYNTHESIS
cDNA synthesis and cloning have become a routine
method with tremendous technological advances and the
development of numerous kits. cDNA libraries can be
established from small amounts of RNA, and rare species
of mRNA can be cloned into a variety of vectors. Careful
thought must be given to the source tissue/sample of
RNA, integrity of RNA, the relative abundance of the
mRNA of interest, the vector, and the method used to
screen the library. The first strand of the cDNA is synthe-
sized using a reverse transcriptase (Figure 9), total RNA
or mRNA and an oligo-dT primer or a random hexamer
(pdN6). The reverse transcriptase enzymes available com-
mercially are from avian or murine RNA, and some have
been engineered to have low RNase H activity (Super-
script, Invitrogen), which increase the yield and the length
of first-strand synthesis. The mRNA is subsequently re-
moved by treatment of RNase H, and synthesis of sec-
ond-strand synthesis is usually catalyzed by DNA poly-
merase 1 and T4 DNA polymerase. These enzymes also

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Table 1
Companies Offering Molecular Biology Tools and Reagents

Product Companies Reagents Web Address

Agilent Lab on a chip www.agilent.com

Ambion RNA products http://www.ambion.com/
BD Biosciences Reporter genes, vectors, cell lines http://www.bdbiosciences.com/clontech/
Bio-Rad Laboratories Restriction enzymes, ligase, gel electrophoresis www.biorad.com
Integrated DNA Technologies Custom oligonucleotide synthesis http://www.idtdna.com/
Invitrogen Vectors, plasmids, enzymes, E. coli cells www.invitrogen.com
New England Biolabs Restriction endonucleases, other enzymes http://www.neb.com/neb/
Promega Prokaryotic, eukaryotic vectors, bioluminescence vectors www.promega.com
Qiagen, Inc. DNA, RNA purification kits www.qiagen.com
Xenogen Optical, bioluminescence imaging equipment www.xenogen.com

Table 2
Useful Measurements and Facts SUMMARY

Molecular weight (average) 649 Daltons (Da) We have discussed the basics of DNA and RNA meth-
of a DNA base pair ods. The protein methods will be included with the re-
Molecular weight (average) 110 Da view on proteomics. Molecular cloning and current proto-
of an amino acid
cols in molecular biology are two excellent manuals rou-
1 Absorbance at 260 nm of 50 ␮g/mL
double-stranded DNA tinely used in laboratories as reference books for various
1 Absorbance at 260 nm of 37 ␮g/mL methods (4,5). There are also web sites that provide tools
single-stranded DNA for analysis, databases, and protocols (6,7). The next two
1 Absorbance at 260 nm of 40 ␮g/mL articles in this series will build up and discuss the large-
single-stranded RNA
1 ␮g/mL of 1 Kb DNA 3 nm fragment ends
scale biology approaches and the disciplines of genomics
fragment and proteomics.

REFERENCES

1. Pandit SD, Bednarski M, Li KC. Review article series. A primer on mo-

remove the protruding single-stranded 3⬘ termini with
their 3⬘3 5⬘ exonucleolytic activity and fill in the re-
cessed 3⬘-hydroxyl ends with their polymerizing activity.
This generates blunt end cDNA molecules, which are then
ligated to linkers containing restriction sites using T4 DNA
ligase. The molecules are cleaved at the restriction site in the
linker and ligated to the vector cut with compatible cohesive
ends, which generates a cDNA library. The ligation mixture
is transformed into a bacterial or phage host depending on
the vector. Such a collection of clones can be propagated
and screened for identification of specific genes using meth-
ods such as nucleic acid hybridization, PCR, or expression
screening using antibodies against the specific protein.
lecular biology for imagers: I. DNA, how does it work? Acad Radiol
2003; 10:1215–1223.
2. Pandit SD, Li KC. Review article series. A primer on molecular biology
for imagers. II. Transcription and gene expression. Acad Radiol 2004;
11:333–344.
3. Pandit SD, Li KC. Review article series. A primer on molecular biology
for imagers: III. Proteins: structure and function. Acad Radiol 2004; 11:
448 – 461.
4. Sambrook J, Russell DW. Molecular cloning: A laboratory manual. 3rd
ed. Plainview, NY: Cold Spring Harbor Laboratory Press; 2001. Also
available at: (www.molecularcloning.com). Accessed March 25, 2004
5. Ausubel AM, Brent R, Kingston RE, et al, eds. Current protocols in mo-
lecular biology. Hoboken, NJ: John Wiley and Sons, Inc, 1998.
6. CMS Molecular Biology Resource. Available at: http://mbcf.dfci.
harvard.edu/cmsmbr/. Accessed 15 March 2004.
7. Computational Molecular Biology at NIH. Available at: http://molbio.
info.nih.gov/molbio/. Accessed 15 March 2004.

S53

A Primer on Molecular Biology for Imagers:
V. Genomics: A Global Approach to the Study of Genes1
Building on our discussion of DNA, RNA, and proteins
(1– 4), this article will introduce genomics and the global
approaches to understanding the molecular biology of the
cell. It is worthwhile to reiterate that genes and their
products do not function independently, but rather they
partake in complex, interconnected pathways and net-
works—molecular systems—that ultimately determine the
phenotype of cells, tissues, organs, and organisms. Every
phenotype, normal or pathological, begins with initiating
molecular events that feed into these pathways and net-
works that eventually determine the fate of a cell. Thus,
to fully understand the molecular biology of a cell—nor-
mal or diseased—scientists must look beyond individual
genes or their products to address the global cellular envi-
ronment responsible for a particular phenotype.
From its birth, genomics is the field of biology that
aims to understand the molecular organization of the en-
tire genome and the products it encodes. Just a few years
ago, the discipline of genomics was merely divided into
two main areas: 1. structural genomics—the area that fo-
cused on the physical characteristics of the genome in-
cluding mapping and sequencing of genes and 2. func-
tional genomics—the area that aimed to understand the
function of genome and its products. In general, the se-
quenced structure of a specific segment of DNA must first
be characterized before the study of its function. Thus the
scope of genomics rapidly transformed upon the comple-
tion of the Human Genome Project (HGP) in April of
2003. Coupled with recent advances in molecular tech-
Reprinted with permission from Acad Radiol 2004; 11:817– 828
1 From the Molecular Imaging Laboratory, Department of Diagnostic Radiol-
ogy, Clinical Center, 10/1N306, National Institute of Health, 9000 Rockville
Pike, Bethesda, MD 20892(K.V., S.D.P., K.C.P.L.). Received and accepted
April 28, 2004. Address correspondence to K.V. e-mail: kvuu@cc.nih.gov
© AUR, 2004
doi:10.1016/j.acra.2004.10.006
S54
Chapter V
Kien Vuu, BS, Sunil D. Pandit, PhD, King C.P. Li, MD, MBA
niques and bioinformatics, the HGP provided the neces-
sary data set to expand new areas of studies such as com-
parative genomics, proteomics, phenomics, pharmacog-
enomics, and toxicogenomics.
In the present day, it is feasible to consider a broader
view of genomics as the study of the genome, its varia-
tion, its products and their dynamic interplay during phys-
iologic, pharmacologic, and pathologic states. Genomic
research has the potential to characterize the molecular
basis of all diseases, an accomplishment that would en-
able the early detection of disease, refine current disease
diagnosis and classification, and expedite the development
of new molecular-targeted therapies. Another endeavor of
genomic research is to identify variability in the genome
and to understand what role these variations play in
health and disease or in the different responses to drug
therapy.
With recent advances in the clinical translation of
genomic research, it is certain that this genomic revolu-
tion will fundamentally change the way future generations
of healthcare professionals of all disciplines practice med-
icine. The purpose of this article is to introduce current
key concepts in genomics and to provide a framework for
radiologists to familiarize themselves with the explosive
changes in medicine in this genomic era. Therefore, this
article will begin by highlighting the early work in the
field with structural genomics; review current technolo-
gies in genomic research such as comparative genomics,
functional genomics, and bioinformatics; and conclude
with a discussion of the future of genomics with a men-
tion of opportunities and challenges for those in the imag-
ing sciences.
EARLY EFFORTS: STRUCTURAL GENOMICS
Much of the early work in genomics focused on struc-
tural genomics. Setting the stage for this work were de-
 Reprinted from Academic Radiology, Vol 11, No 7, July 2004 A GLOBAL APPROACH TO THE STUDY OF GENES

Figure 1. Basic organization of the human genome. This chart represents the basic schema of the human genome based on early ge-
netic efforts. Since this work was completed prior to understanding the full structure and sequence of the genome, it is only an estimate
of what the genome contains based on studies of isolated genome components. To increase the resolution and accuracy of this organi-
zation, the genome must be completely mapped and sequenced. (Reproduced with permission from Strachan and Read. Human Molec-
ular Genetics 2. 2nd ed. Oxford, UK: BIOS Scientific Publishers Ltd; 1999.)

velopments in genetics that described a general organiza-
tion of the human genome (Figure 1). The discipline of
structural genomics aims to build on this work by charac-
terizing the structure and sequence of model genomes.
The rationale for this effort was that with this informa-
tion, it would be possible to identify new genes, make
predictions of all encoded proteins, and to understand and
characterize variation within a species. Also by compar-
ing genomes between species, it would be possible to
understand how genomes are remodeled during evolution
and identify conserved, functionally important sequence
motifs. In essence, scientists knew that to fully understand
the function of the genome, the structure of the genome
must first be characterized. After much scientific debate
and dialogue throughout the 1980’s, the National Insti-
tutes of Health along with the Department of Energy
launched the Human Genome Project in 1990 with the
goal of sequencing the genomes of the human and several
model organisms. The concept of genome sequencing was
only conceivable after key discoveries and technologies
were established. Sanger sequencing (4) allowed the se-
quencing of small DNA fragments of up to 700 – 800 base
pairs (bp) in length. The constraint of these fragment
sizes is due to limitation in polyacrylamide gel resolution.
This sequencing method was exponentially expedited by
the use of fluorescent dideoxynucleotides and automated
sequencing technology developed by Leroy Hood. How-
ever, since automated sequencers could only sequence
700 – 800 bp fragments at a time, detailed genetic and
physical maps (Figure 2) were necessary to piece together
these sequenced fragments. In a step-by-step fashion, the
discovery of polymorphisms and the advances in genetic
and physical mapping played crucial roles in the struc-
tural characterization and sequencing of large genomes.
Polymorphisms
By the late 1980’s, the discovery of polymorphisms
increased our understanding of genome variation and pro-
vided a tool for genome mapping. Though intra-species
genomes are virtually identical, most individual genomes

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Figure 2. Combined Genetic and Physical Map of a Chromosome. (A) The enormous quantity of DNA contained in each chromosome
can be mapped by the step-wise identification of molecular markers with increasing resolution. Large markers such as genes can first be
the landmarks of these maps. These and other markers can be used to help order contiguous chunks of DNA that can be cloned into
vectors such as cosmids, BACs, or YACs (4). Further identification of higher-resolution markers by both genetic and physical mapping
methods can then be used to piece together short sequenced fragments of DNA. Please refer to text for details. (B) A detailed map of
chromosome 17 displaying locations of molecular markers with the isolation of the BRCA1 gene flanked between specific markers.
(Adapted with permission from Griffiths AJF, Miller JH, Suzuki D, Lewontin RC, Gelbart WM. Introduction to Genetic Analysis. 7th ed.
New York, NY: WH Freeman & Co; 1999 and New York Times Graphics © 1994).

within a species will have their own unique sequence.
Polymorphisms are the existence of variants—sequence
variants, allelic variants, and phenotypic variants—within
a population. Two main types of polymorphisms exist:
Single Nucleotide Polymorphisms (SNPs) and Simple
Sequence Length Polymorphisms (SSLPs). SNPs are spe-
cific positions in the genome where individuals differ in a
single nucleotide. By contrast, SSLPs are repeat se-
quences that display length variation. They could be fur-
ther classified into minisatellites (VNTRs) and microsatel-
lites (STRs) depending on size of their repeat units. Be-
cause intra-species genomes are nearly identical, these
polymorphisms provide good markers or landmarks used
in the mapping of genomes.
Genetic and Physical Mapping
Another pivotal discovery prior to genome sequencing
was genome mapping. A map of the genome, like any
geographic map, provides important information about the
location and relative positions of landmarks. Instead of
lakes and cities separated by states and highways, maps
of genomes reveal locations of genes or specific DNA

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 Reprinted from Academic Radiology, Vol 11, No 7, July 2004
sequences separated by chromosomes and nucleotides.
Two types of maps exist: genetic and physical maps—the
difference between the two lie in the methods used in
their production. Genetic maps utilize genetic techniques
in their creation, while physical maps employ molecular
biology techniques (i.e. physically manipulate DNA mole-
cules) in their construction.
Genetic mapping is a technique used to determine the
genetic distance between two markers. Built on the funda-
mentals of linkage and recombination, the genetic dis-
tance between two markers can be assigned by calculating
the frequency that two markers recombine after meiosis—
the recombination frequency. The markers used in genetic
mapping must have at least one variant to monitor a re-
combination event. Early mapping studies used genes as
markers; however, mapping the genome was not possible
because of the paucity of known genes. With the discov-
ery of polymorphisms that were distributed throughout the
genome, whole genome genetic mapping became feasible
using SNPs, SSLPs, and RFLPs (Restriction Fragment
Length Polymorphism) as markers. This form of linkage
analysis is used in pedigree or cross-breeding studies to
map out the order of genes and markers within a chromo-
some. In fact, the positional cloning of the Huntington’s
and CFTR (cystic fibrosis transmembrane conductor regu-
lator) genes were made possible by genetically mapping
out markers that were closely linked to these genes, and
then analyzing the regions approximate to markers to
identify the gene.
Due to the limited accuracy and variable resolution of
genetic mapping, alternative mapping procedures have
been developed to validate and supplement genetic maps.
These physical mapping techniques include restriction
mapping, fluorescent in situ hybridization (FISH), and
sequence tagged site (STS) mapping. Restriction mapping
involves a set of DNA digest reactions with the use of
two or more restriction enzymes to construct a map of
different restriction sites (Figure 3A). By contrast, FISH
utilizes fluorescent probes to hybridize to and thus map
and localize specific DNA sequences within the genome
(Figure 3B). Though restriction mapping and FISH are
useful mapping techniques, only STS mapping allows the
rapid, high-resolution mapping of a large genome. There
are two main components to STS mapping: 1) STS sites
and 2) a collection of redundant and random, overlapping
DNA fragments of a chromosome or genome (Figure 4).
STS sites are any unique sequences of DNA about 100 –
500 bp in length that would appear only once in a chro-
mosome/genome. By assigning multiple STS sites across
A GLOBAL APPROACH TO THE STUDY OF GENES
a chromosome/genome, the fragment collection can be
screened for STS sites using the polymerase chain reac-
tion and STS flanked primers. The data generated allows
computers to align fragments via their STS sites and also
determine the distance between STSs using linkage analy-
sis. The result of this process is the generation of a highly
resolved physical map.
Genome Sequencing
With the advances in DNA cloning, mapping, and se-
quencing in place by the late 1980s, the task of sequenc-
ing the human genome was then feasible. Although dif-
ferent methods were taken to sequence the genome, the
basic approach was very similar. The genome could be
broken up into many overlapping fragments of 500 –700
bp each. With genetic and physical maps of these frag-
ments, each fragment can be individually sequenced via
the Sanger method and then reconstructed to give a com-
plete genome sequence map.
The completion of Human Genome Project is indeed a
crowning achievement, however, the major challenge of
understanding the contents and functions of the genome
still remain. The completed sequence of the genome is
similar to a Choose Your Own Adventure book in a dif-
ferent language. We know that this book is not meant to
be read from front to back—that the paths taken from
reading different pages will lead one to a different fate.
Sequencing of the genome is similar to revealing all the
letters of this book in the correct sequence, but since it is
in a different language, there is still much work to dis-
cover the words and paths one could take. This is analo-
gous to the task of looking for and understanding new
genes or other functional regions of the genome and deci-
phering the molecular networks that each cell operates on.
The next sections of this article will focus on current and
future directions of this discovery process.
CURRENT TOPICS: COMPARATIVE
GENOMICS, FUNCTIONAL GENOMICS,
BIOINFORMATICS, AND TRANSLATIONAL
GENOMICS
Developed alongside the technologies leading to ge-
nome sequencing were advances in experimental methods
and bioinformatics. These parallel processes iteratively
interacted in such a way as to exponentially expand the
field of genomics. The completed sequences of model
genomes have provided the fundamental data set and
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 VUU ET AL Reprinted from Academic Radiology, Vol 11, No 7, July 2004

Figure 3. Physical Mapping Techniques. (A) Restriction mapping. A 6.2 kb DNA sample is digested in 6 separate reactions involving
individual and combined enzymes. The single digest reactions (Bgl II, Bam HI, and Pst 1) will yield variable-sized DNA fragments that
give information about the number of restrictions sites within a DNA molecule, while the fragments produced from the combined reaction
will allow the positioning of each restriction site relative to each other. A map of the Bgl II, Bam HI, and Pst 1 restriction sites can be
constructed by analyzing the sizes of the restriction fragments produced from each digest reaction. (B) FISH. A specific DNA sequence
can be localized among a collection of fixed chromosomes by exposing the chromosomes to fluorescent probes of that DNA sequence
and observing where they have hybridized. (Adapted with permission from Brown TA. Genomes 2. 2nd ed. Oxford, UK: BIOS Scientific
Publishers Ltd; 2002. and Strachan and Read. Human Molecular Genetics 2. 2nd ed. Oxford, UK: BIOS Scientific Publishers Ltd; 1999.)

groundwork for current genomics efforts to build on. This
next section focuses our current status in genomics, high-
lighting comparative genomics, functional genomics,
bioinformatics, and translational advances of genomics in
clinical medicine.
Comparative Genomics
One of the original goals of the Human Genome
Project (HGP) was not only to sequence the human ge-
nome, but also the genomes of several model organisms.
With the completion of the HGP, the human, rat, mouse,
drosophila, yeast, and other model genomes have been
completely sequenced. These genome sequences are now
available in public databases and are the foundation for
large-scale comparative genomic analysis. Comparing the
genomes of model organisms, from simple to more com-
plex, proves to be a good method in understanding the
function of the genome as well as the evolution of ge-
nomes. The main assumption underlying comparative
genomics is that the genomes under study have a com-
mon ancestor and that each base pair in those genomes
are derived from this common ancestor and the action of
evolution. Thus highly conserved sequences are consid-
ered functionally important and will aid in gene and regu-
latory region predictions. Comparing genomes also aids in
gene mapping and evolutionary studies. Early work in
comparative genomics has shown that many organisms
display a great deal of synteny (conserved gene order).
Thus, it would be possible to use map information from
one genome to locate genes in another; and moreover,

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Figure 4. Sequence tagged site mapping. (A) A redundant collection of overlapping

DNA fragments spans the entire chromosome with each site represented about 5
times. (B) Once STS sites are assigned, the fragment collection is screened by PCR to
see which fragments contain which STSs. (C) Computer-aided technology can aid in
aligning STS sites as well as determine the distance between STS sites to construct a
physical map. (Adapted with permission from Griffiths AJF, Miller JH, Suzuki D, Le-
wontin RC, Gelbart WM. Introduction to Genetic Analysis, 7th ed. New York, NY: WH
Freeman & Co; 1999.)

synteneic regions from different genomes can be aligned

by computer programs to assess evolutionary divergence.
Comparative genomics prove to be a powerful tool in
understanding genomes, and currently, more genomes are
being sequenced to increase its analytical power.

Functional Genomics
The availability of sequence data allows scientists to
study the function of that sequence. Thus with the large
data sets produced by the HGP explodes the field of func-
tional genomics, an area of genomics aimed at studying
the function of the genome and its products. The use of
this term has been recently popularized by DNA microar- Figure 5. The six reading frames of a duplex DNA molecule.
ray technology; however, scientists have been studying Each strand of DNA has three reading frames—totaling six for a
duplex molecule. Each of these reading frames is read as a con-
the functions of segments of genome sequences from the tinuous series of triplets (codons) and can be scanned for open
birth of genomics. reading frames—stretches of DNA within a reading frame that be-
Early and continued work in functional genomics in- gin with a start codon and end with a termination codon.

volved the discovery of new genes and the study of the

function of individual genes. With sequence information,
ORF (open reading frame) analysis can be used for gene ular DNA sequence for stretches of DNA beginning with
discovery. This technique involves using a computer pro- the universal start codon and terminating with a stop
gram to scan all six reading frames (Figure 5) in a partic- codon. These DNA segments are potential genes that can

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 VUU ET AL Reprinted from Academic Radiology, Vol 11, No 7, July 2004

Figure 6. Inactivating gene function. (A) RNA interference. Double-stranded RNA can be introduced into cells and will subsequently be
cleaved into siRNAs by the RNA nuclease enzyme DICER. The siRNAs will bind to the target mRNA and lead to its destruction. (B) Ho-
mologous recombination. Homologous regions of a vector DNA sequence and a target gene can recombine and disrupt or “knock-out”
the target gene. (Reproduced with permission from Brown TA. Genomes 2. 2nd ed. Oxford, UK: BIOS Scientific Publishers Ltd; 2002.)

be further analyzed for function. For example, these
ORFs can be analyzed for conserved sequence motifs or
homology using computers to clue scientists into the
functional regions in that particular segment. Other meth-
ods for studying the function of individual genes involve
experimental methods that inactivate or enhance the ex-
pression of a particular gene and predicting their function
by observing the phenotype of this manipulation. Major
techniques used for gene inactivation include RNA inter-
ference and homologous recombination. In RNA interfer-
ence, double-stranded RNA molecules are introduced into
cells and are cleaved into siRNAs (or small interfereing
RNAs) whose sequences match that of the mRNA being
targeted. The siRNAs can bind to and lead to the destruc-
tion of the target mRNA—thereby functionally silencing
the expression of a target gene (Figure 6A). By contrast,
homologous recombination can destroy gene function at
the DNA level by introducing a homologous DNA se-
quence that could insert into and “knock-out” the gene of
interest (Figure 6B). On the other hand, genes can also be
induced to overexpress its function. Inserting a highly
active promoter next to a gene of interest, or cloning in
multiple copies of a gene into the genome could act to
increase the expression of the gene under study.
In contrast to the techniques studying single genes,
microarray technology allows the high-throughput, global
analysis of the genome and its products. The basic con-
cept behind microarrays is the “DNA chip” in which
DNA sequences are attached—arrayed into spots— onto a
solid support, such as a glass slide. Labeled test samples
(targets) taken from cells or tissues can then be allowed
to hybridize with the arrayed spots (probes) on the mi-
croarray, and this binding can be quantified. In essence,
the array acts as a pool of possible sequences to screen or
probe a particular sample for. Depending on what se-
quences are arrayed on the chip, different information can
be obtained. For example, whole genome cDNA chips
can be used to measure the gene expression profile of a
particular sample by quantifying the hybridization of
cDNA targets prepared from that sample; or, sequences
with common polymorphisms or mutations can be arrayed
on chips to probe for their presence in a given sample.
With the completion of the HGP, it is now possible to
construct a variety of arrays useful for comparative
genomic hybridization, gene identification, and screening
for specific sequences or nucleotides such as promoters,
polymorphisms, and mutations.
The most popular application in microarray technology
is comparing gene expression profiles between two sam-
ples, and it will be used as a model to discuss two popu-
lar microarray systems. Recall that the probes attached to
the solid array surface determine the utility of that partic-
ular array. Since cDNAs and ESTs (expressed sequence
tags) are considered surrogates for gene expression, they
could be used as probes to monitor this biologic event in
cellular or tissue samples. The two systems for studying
gene expression are cDNA microarrays and oligochips
(GeneChip as commercialized by Affeymetrix Inc). One
main difference between these systems lies in the synthe-
sis of these chips. The cDNA arrays are made by spotting

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 Reprinted from Academic Radiology, Vol 11, No 7, July 2004
multiple copies of cDNAs onto a solid support. By con-
trast, oligochips are designed by assembling 15–20 bp
oligonucleotide sequences directly onto the chip surface
with the use of photolithography and DNA chemical anal-
ysis. These oligonucleotides are unique sequences that
represent all the ESTs in the genome. Though these two
systems differ slightly in other methodical aspects, the
main components of a cDNA microarray or oligochip
experiment are essentially the same. They include:
1. Preparation of targets— extracting/preparing nu-
cleic acids from biological samples and labeling
them with a detection agent (fluorescent dyes). For
gene expression analysis, RNA is isolated from a
test sample, converted into cDNA, and then labeled
with a detection agent.
2. Hybridization to probes—incubating labeled targets
to the probes on the microarray.
3. Scanning the chip— using confocal microscopy and
computer-assisted reading to determine the signal
intensity of labeled targets at each probe spot on the
microarray. This value quantifies the relative
amounts of target hybridized to each specific probe.
4. Analyzing the data—transforming the large data sets
from these experiments into biologically useful in-
formation.
By measuring the signal intensity at each probe spot on
the array, the amount of the specific target sequence in
the sample can be quantified. For these cDNA and oligo
chips, a quantified gene expression profile for each sam-
ple can be obtained; however, these experiments are sel-
dom performed with single samples. Rather, gene expres-
sion is usually expressed in relation to a reference sam-
ple. In cDNA arrays, two sets of differently labeled
targets from two samples are allowed to hybridize on the
same chip, and the relative intensities of the different la-
bels on each probe determine the relative expression of
each gene. For oligo-arrays, only one target sample is
hybridized per array, and the relative expression is deter-
mined by comparing a sample array to a reference array
(Figure 7). Comparing gene expression using microarrays
is a powerful tool widely used in genomic research today.
For example, comparing “normal” samples to “diseased”
samples can identify new candidate genes or pathological
pathways involved in disease. Also, by comparing hetero-
geneous samples from a population of patients with the
“same disease” or clinical diagnosis, new molecular
classes of disease can be identified which will be useful
A GLOBAL APPROACH TO THE STUDY OF GENES
in disease reclassification or selecting treatment options.
Expression profiles of known disease classes can then be
used to diagnose and classify unknown samples.
Bioinformatics
From our review of structural, comparative, and func-
tional genomics, it is clear that advanced bioinformatics
was necessary for the advancement of genomics. By defi-
nition, bioinformatics is the discipline that integrates in-
formation science, computer science, mathematics, and
associated technologies into the field of molecular biol-
ogy. Every aspect of genomics research we have dis-
cussed thus far utilizes these technologies to store, orga-
nize, and analyze the large data sets produced from such
studies. Since this information can be made public to sci-
entists around the world, research efforts can expand
without redundancy— e.g. a researcher does not have to
sequence several genomes on their own before doing a
comparative study. In general, bioinformatics is divided
into two main genres: databases and tools. Databases
store the vast amount of data generated from experiments
and many are made accessible to the public. For example,
there are major databases for genome and nucleotide se-
quences: GenBank (5), EMBL (6), and DNA Data Bank
of Japan (7). There are also other databases that store
EST or cDNA information, amino acid sequences, and
data regarding transcription factors, signaling and infor-
mation pathways. From these databases, scientists can
access vast amounts of information and analyze their own
data using sophisticated bioinformatics tools. Tools are
computer programs and algorithms used to organize and
analyze data. For example, a scientist with a particular
DNA sequence could query the databases to see which
organisms contain this sequence, or where this sequence
is located in relation to other genes. Other bioinformatics
tools include programs to draw maps or join overlapping
fragments of DNA sequences, search for restriction sites,
compare DNA or amino acid sequences, align genomes
for comparative studies, or predict function of DNA or
amino acid sequences—this just being a curtailed list of
the many tools in bioinformatics. The developing field of
phenomics aims to link gene expression, proteomic,
genomic, and clinical data to produce programs that pre-
dict phenotype from a given molecular profile.
Shared databases and tools are key elements that could
expand the productivity of any research group. This sci-
entific exchange could also be used to reach a scientific
endeavor or goal—the center of such exchange being a
consortium. Two archetypal examples include the IMAGE
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 VUU ET AL Reprinted from Academic Radiology, Vol 11, No 7, July 2004

Figure 7. cDNA Arrays versus oligochips. The upper portion of the figure shows the difference in the construction of the arrays. cDNA
(left) arrays are spotted, while oligonucleotides synthesized on the oligochips are synthesized in situ. The bottom portion of the figure
demonstrates the target preparation. Both sample and reference targets in cDNA chip experiments hybridize to the same cDNA array
(middle left), while sample and reference targets used in oligochip experiments are hybridized to two separate oligochips (middle right).
Please refer to text for details. (Reprinted with permission from Schultz and Downward. Nature Cell Biology. Vol 3. E190-5. MacMillan
Magazines Ltd.)

(8) and CGAP (9) consortiums. The IMAGE (Integrated
Molecular Analysis of Genomes and their Expression)
consortium was founded in 1993 by several academic
groups with the vision of better understanding the human
genome. It provides sequence, map, and expression data
of several model organisms as well as bioinformatics
tools. The IMAGE consortium also shares high-quality,
full-length cDNA arrays for other scientists to use in their
own projects. Also in the collaborative spirit is the CGAP
(Cancer Genome Anatomy Project) consortium with the
goal of determining “the gene expression profiles of nor-
mal, precancer, and cancer cells, leading eventually to

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improved detection, diagnosis, and treatment for the pa-
tient.” To meet this goal, they have established one of the
world largest public scientific databases and enabled sci-
entists anywhere access to the latest bioinformatics tools
and teaching materials on methods and reagents. These
examples foreshadow the type of interplay and collabora-
tive team science that future genomics efforts must adapt
to most efficiently answer the many questions present
today in molecular biology.
Translational Genomics
As the resolving power to study disease mechanisms
becomes more molecular, medical professionals can
increase their clinical precision in diagnosing, monitor-
ing, and treating patients. A great deal of genomic re-
search has already been translated into the practice of
medicine and drug discovery. The identification of new
candidate disease-associated genes have allowed the
pre-symptomatic identification of high-risk patients;
screening for BRCA1 or BRCA2 gene mutations in
females with a family history of breast cancer aids
physicians’ decisions on how to monitor a patient. Un-
derstanding the molecular basis of disease can reclas-
sify previous disease diagnoses; microarray data en-
abled the subclassification of diffuse large B-cell lym-
phoma (DLBCL) into two molecularly distinct groups
each with a different set of treatment options (10).
Also the identification of disease genes or associated
proteins mark new targets for drug discovery; the dis-
covery of the disease defining 9:22 chromosomal trans-
location in Chronic Myelogenous Leukemia (CML) and
its associated bcr-abl protein led to the development of
Gleevec, a drug that has drastically improved the prog-
nosis in patients with CML (11). Genomic data can
also guide precision drug delivery; for breast cancer
patients, the drug tamoxifen(a chemotherapeutic and
estrogen receptor partial agonist/antagonist) is only
given after establishing that the patient’s cancer cells
express estrogen receptors, and dosing of this drug can
be adjusted according to the expression level of those
receptors (12). This form of clinical accuracy is now
known in the medical community as personalized med-
icine. While these are just a few of the many transla-
tional examples of genomics, it is certain that a
genomic revolution will fundamentally change the way
future generations of health care professionals of all
disciplines practice medicine.
A GLOBAL APPROACH TO THE STUDY OF GENES
FUTURE OF GENOMICS
As recent advances in the clinical translation of
genomic research continue to explode, it is appropriate to
discuss the future directions of genomics to prepare radi-
ologists for the type of medicine that is soon to come. As
discussed earlier, the completion of the Human Genome
Project has provided a springboard for many other areas
of genomics to expand. Upon its completion, Francis Col-
lins et al (13) released a pivotal article in Nature, “A vi-
sion for the future of genomics research: A blueprint for
the genomic era.” In the article, this vision is formulated
into three major themes: 1. Genomics to Biology— eluci-
dating the structure and function of genomes, 2. Genom-
ics to Health—translating genome-based knowledge into
health benefits, and 3. Genomics to Society—promoting
the use of genomics to maximize benefits and minimize
harms. This next section will highlight what to expect in
the near future of genomics.
Further Understanding the Molecular Biology of
the Cell
Much effort is currently being applied to understanding
all the biological aspects of the genome. The work to
comprehensively identify all the structural and functional
elements of the genome is under way, and the tools of
comparative and functional genomics along with emerg-
ing technologies will facilitate this process. Another im-
portant biological endeavor is to understand the organiza-
tion of genetic networks and protein pathways and how
specific molecular profiles will contribute to specific cel-
lular and organismal phenotypes. This venture will re-
quire the global understanding of molecular systems at
several levels. Microarrays have offered the ability to as-
sess the molecular complexity of phenotypes at the ge-
nome level, while the field of proteomics (addressed in
the next article of this series) aims to characterize these
molecular dynamics at the protein level. As more technol-
ogies advance to fill in the gaps in our understanding of
all levels of molecular intricacy, the field of phenomics
will emerge by integrating these multi-level data sets to
predict phenotype. Finally, an international concerted ef-
fort to understand the heritable variation in genomes is
under way. Formed in 2002, the International HapMap
project aims to catalogue all common polymorphisms in
the human population and understand how these varia-
tions are inherited. Given that each human being shares a
virtually identical genome sequence, the HapMap project
will play a crucial role in explaining differences in indi-
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 VUU ET AL
viduals’ health and disease susceptibility, drug responses,
or other phenotypes.
Applying Genome Biology to Health Benefits
As the biological aspects of genomes are being re-
vealed, medical professionals will aspire to translate new
found ideas into clinical utility. We have already dis-
cussed examples of genomics guiding pre-symptomatic
disease detection, reclassification of diseases based on
detailed molecular characterization, and personalized de-
livery of drugs based on molecular profiles of the patient.
With continued progress in the HapMap project, scientists
in the area of pharmogenomics and toxicogenomics can
begin to predict the beneficial and adverse reactions to
drugs based on a patient’s genomic profile. Further
genomic studies will reveal more molecular targets in
different disease processes, and small molecular libraries
are currently being used for the high-throughput screening
for diagnostic or therapeutic ligands for these specific
targets. As the understanding of genomes becomes more
complete, one can expect more genome based approaches
to diagnostics and therapeutics. As we will later discuss,
such approaches will include molecular imaging.
Social Implications of Genomic Research
The study of genomes will ultimately answer the fun-
damental questions of mankind’s existence, reveal the
sources of differences in the human race, and predict
nearly any phenotype given a molecular profile. Thus, the
formulation and implementation of social policies are nec-
essary to protect and uphold the values and ethics of our
society. The Ethical, Legal, and Social Issues (ELSI) pro-
gram—the largest bioethics program ever assembled—
was conceived together with the HGP to address such
issues encountered during the advancement of genomics.
After the announcement of completion of the HGP, the
US Senate passed the Genetic Information Nondiscrimina-
tion Act of 2003 to protect citizens against genetic dis-
crimination. Future challenges include understanding the
consequences of revealing the relationships between
genomics, race, and ethnicity or uncovering the genomic
influences on human traits and behaviors.
SUMMARY AND DISCUSSION
The fundamental principle behind global approaches in
molecular biology is that the fate of each cell is the result
of complex networks and pathways of many genes and
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Reprinted from Academic Radiology, Vol 11, No 7, July 2004
their products. Thus in order to fully understand the pro-
cesses leading to any cellular or organismal pheno-
type— be it physiological or pathological—research ef-
forts must focus on observing the precise molecular inter-
actions involving specific amounts of genes and gene
products. Through the groundwork established by the Hu-
man Genome Project, the global study of genes and their
products is now widely in practice. The molecular resolu-
tion of disease characterization established by initial ef-
forts of genomic research has rapidly found utility in clin-
ical medicine. With time, we can only expect that the
parallel disciplines of proteomics and phenomics along
with advances in bioinformatics will enhance the preci-
sion of the molecular characterization of phenotypes, en-
abling the elucidation of molecular pathways to every
disease. It is clear that we are now in the genomic era of
science and the formative years of molecular medicine.
There is no question that molecular medicine will
transform the way medicine is being practiced today. No
longer will the aim of physicians be to assess disease
based on anatomic or physiologic aberrations, but to seek
out the molecular events that are responsible for such ab-
normalities. Thus, for those in the imaging sciences, mere
morphologic information will no longer be a sufficient
means of patient assessment as the demand for molecular
resolution becomes greater. Fortunately, molecular medi-
cine affords many opportunities and imaging will play
an integral role in its practice. As described by Collins et
al, (13) part of the vision of the future of genomics re-
search will include the development of in vivo imaging
methods that allow noninvasive molecular phenotyping.
Many advances, spearheaded by the early work of the
National Cancer Institute, have already demonstrated the
ability to use imaging to characterize and measure biolog-
ical processes at the cellular and molecular level, and this
discipline of molecular imaging is now expanding expo-
nentially. As the specialty of radiology embraces molecu-
lar medicine, it is easy to foresee that imaging will be on
the forefront in guiding the early detection of disease,
precision diagnoses, and delivery of personalized drug
treatments. The challenge in this process, however, lies in
the recruitment of molecular and cellular biologists, phys-
icists, and chemists into radiology to work in synergy to
advance the field of molecular imaging. Furthermore, it
will be necessary to train the next wave of radiologists to
be proficient in the molecular sciences that are rapidly
revolutionizing medicine. The subsequent articles in this
series will discuss further technology in the molecular
study of cells, imaging contrast, and hardware develop-
 Reprinted from Academic Radiology, Vol 11, No 7, July 2004
ments that are advancing molecular imaging methods and
the role of the radiologist in this new era of molecular
medicine.
GLOSSARY
cDNA (copy DNA): a double-stranded DNA copy of
an mRNA molecule synthesized by the enzyme reverse
transcriptase
ESTs (expressed sequenced tags): a short, partial
cDNA sequence used to probe genes or gene products
Knock-out: experimental mutation or deletion of gene
Linkage: the tendency for genes to be inherited to-
gether based on their physical proximity to each other on
the same chromosome
Open Reading Frame: a sequence of DNA containing
a series of codons beginning with a start codon and end-
ing with a termination codon
Pharmacogenomics: the area of science aimed at un-
derstanding the genomic contributions to drug responses
Phenomics: the area of science focused on understand-
ing the molecular mechanisms behind phenotypes
Photolithography: an experimental technique that uses
pulses of light to construct an oligonucleotide sequence
Positional Cloning: a method using map information
of a gene to obtain a clone of that gene
Reading Frame: a series of triplet codons in a DNA
sequence. There are 3 reading frames per strand of DNA
and 6 for a double-stranded DNA molecule.
Recombination: a DNA rearrangement event involv-
ing the physical breakage of homologous chromosomes
and exchange of their homologous DNA segments
RFLP (Restriction Fragment Length Polymor-
phism): a DNA restriction fragment that is variable in
size among a population due to the presence of polymor-
phic (variable) restriction sites in that population
STRs (Short Tandem Repeat): synonymous to micro-
satellite; tandem copies of dinucleotides or trinucleotides
whose copy number varies among individuals in a popu-
lation
Toxicogenomics: an area of science aimed at under-
standing the genomic contributions to adverse reactions to
drugs and other chemicals
A GLOBAL APPROACH TO THE STUDY OF GENES
VNTRs (Variable Number Tandem Repeat): similar
to STRs except VNTRs have larger (⬃25 bp) repeat units
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imagers: I. DNA, how does it work? Acad Radiol 2003; 10:1215–1223.
2. Pandit SD, Li KC. A primer on molecular biology for imagers: II. Tran-
scription and gene expression. Acad Radiol 2004; 11:333–334.
3. Pandit SD, Li KC. A primer on molecular biology for imagers: III.
Proteins: structure and function. Acad Radiol 2004; 11:448 – 461.
4. Pandit SD and , Li KC. A primer on molecular biology for imagers: IV.
Concepts and basic methods in molecular biology. Acad Radiol 2004;
in press.
5. GenBank. http://www.ncbi.nlm.nih.gov/GenBank. Accessed April 5,
2004.
6. European Bioinformatics Institute. http://www.ebi.ac.uk. Accessed
April 5, 2004.
7. DNA Database of Japan. http://www.ddbj.nig.ac.jp/fromddbj-e.html.
Accessed April 5, 2004.
8. The IMAGE consortium. http://image.llnl.gov. Accessed April 5, 2004.
9. The Cancer Genome Anatomy Project. http://cgap.nci.nih.gov. Ac-
cessed April 5, 2004.
10. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large
B-cell lymphoma identified by gene expression profiling. Nature 2000;
403:503–511.
11. O’Dwyer ME, Mauro MJ, Druker BJ. STI571 as a targeted therapy for
CML. Cancer Invest 2003; 21(3):429 –38.
12. Bennink RJ, van Tienhoven G, Rijks LJ, Noorduyn AL, Janssen AG,
Sloof GW. In vivo prediction of response to antiestrogen treatment in
estrogen receptor-positive breast cancer. J Nucl Med 2004; 45:1–7.
13. Collins FS, Green ED, Guttmacher AE, Guyer MS. A vision for the fu-
ture of genomics research: A blueprint for the genomic era. Nature
2003; 422(6934):835– 847.
ADDITIONAL REFERENCE MATERIALS
Brown TA. Genomes. 2nd ed. Oxford, UK: BIOS Sci-
entific Publishers Ltd; 2002.
Griffiths AJF, Miller JH, Suzuki D, Lewontin RC,
Gelbart WM. Introduction to genetic analysis. 7th ed.
New York, NY: WH Freeman & Co; 1999.
Strachan T, Read A. Human molecular genetics. 2nd
ed. Oxford, UK: BIOS Scientific Publishers Ltd; 1999.
Brown S. Essentials of medical genomics. Hoboken,
NJ: John Wiley and Sons; 2003.
Primer on Medical Genomics: Parts I–X. Mayo Clinic
Proceedings 2002;77:773–782, 785– 808, 927–940, 1185–
1196 –(78) 2003;78:57– 64, 307–317, 580 –587, 846 – 857,
1098 –1109, 1370 –1383.
Ureta-Vidal A, Ettwiller L, Birney E. Comparative
genomics: Genome-wide analysis in metazoan eu-
rkaryotes. Nature Reviews: Genetics 2003;4:251 –262.
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A Primer on Molecular Biology for Imagers:
VI. Proteomics: The Large-Scale Study of Proteins1
Joseph T. Azok, BS, Sunil D. Pandit, PhD, King C.P. Li, MD, MBA
Building on genomics and the success of the Human Ge-
nome Project (HGP), this article will introduce the field
of proteomics and the potential it has for making substan-
tial contributions to biomedical and clinical research. In-
formation gained from the HGP has given us a detailed
“blueprint” of the 25,000 –30,000 genes contained within
the human genome. Using these data, significant advances
have been made in our understanding of genes and their
regulation. However, the downstream effectors of genes,
namely proteins, are thought to be more interesting and to
hold the most clinical significance. Proteins are involved
in nearly all biologic activities, are central to most disease
processes, and are the targets of the vast majority of
drugs. Proteomics has the potential to allow for the much
broader understanding of how proteins function together
at the molecular level, which hopefully will translate into
a better understanding of disease processes and improved
clinical therapies.
Proteomics is defined as the study of all the proteins
within an organism. An all-encompassing term, proteom-
ics attempts to describe the identity, quantity, three-di-
mensional structure, interactions, posttranslational modifi-
cations, and functions of all the proteins expressed within
a particular location at any one point in time. Unlike the
genomic sequence, which is essentially static and identi-
cal within all cells, the proteome is unique to each cell
and constantly changes with each physiologic and patho-
logic process. The emerging field of proteomics seeks to
make sense of the variation in protein abundance and ac-
Reprinted with permission from Acad Radiol 2004; 11:940 –950
1 From the Molecular Imaging Laboratory, Department of Diagnostic Radiol-
ogy, Clinical Center, 10/1N306, National Institutes of Health, 9000 Rockville
Pike, Bethesda, MD 20892. Address correspondence to K.C.P.L.
© AUR, 2004
doi:10.1016/j.acra.2004.10.007
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Chapter VI
tivity during different biologic states and describe how
proteins function together on an unprecedented scale—at
the systems-level.
The sequencing of the genome combined with the de-
velopment of DNA microarrays made possible gene ex-
pression profiling, the identification of characteristic ex-
pression patterns through the analysis of thousands of
genes. This method has been instrumental for understand-
ing molecular processes, elucidating drug-target interac-
tions, and has been helpful in clinical diagnosis. How-
ever, the major problem with genomic-based experiments
is their imperfect correlation (estimated by some studies
at only 0.4) between gene activity and protein abundance.
Consequently, genomic experimental results must be con-
firmed by laborious protein assays. Studying proteins di-
rectly through proteomic experiments has several advan-
tages over genomic studies, but also some important limi-
tations.
The field of proteomics today can be compared and
contrasted with the field of genomics 20 years ago, before
the sequencing of the human genome. At that time, rela-
tively primitive genetic technologies existed, and there
was debate as to whether its goals would ever be met.
However, after only a few decades of work, the sequenc-
ing’s goals were achieved. Similarly, the field of proteom-
ics is in its infancy with relatively primitive technology
but where significant contributions continue at a rapidly
progressing rate. In the past few years, major technologic
advances coupled with improvements in bioinformatics
and the completion of the HGP have made high-through-
put proteomic studies possible and led the scientific com-
munity away from isolated studies of single proteins to
larger, more complex projects that attempt to analyze
hundreds or even thousands of proteins simultaneously. In
addition to providing scientists with a more detailed un-
derstanding of fundamental biologic processes, proteomics
has the potential to make significant contributions to clini-
 Reprinted from Academic Radiology, Vol 11, No 8, August 2004 PROTEOMICS: THE LARGE-SCALE STUDY OF PROTEINS
cal medicine, especially in the identification of novel dis-
ease markers and accelerated drug development.
Today, many large-scale, high-throughput proteomic
studies are under way. Guided by the Human Proteome
Organization (HUPO), which was formed by an inter-
national consortium of scientists to coordinate large
proteomic initiatives, its major initiatives are to study
the proteomes of human plasma, brain, and liver, to
develop antibodies against thousands of human pro-
teins, and to standardize the analysis of proteomic
data (1). To date, proteomic strategies have been ap-
plied extensively to study the proteomes of many or-
ganisms, including Helicobacter pylori, Mycobacterium
tuberculosis, Saccharomyces cerevisiae (yeast),
Plasmodium falciparum (responsible for malaria trans-
mission), and several human subproteomes (such as
serum, mitochondria, cardiac muscle, cell nuclei). In
only 5 years, the number of proteomic publications in
the PubMed database grew from 19 in 1998 to 1,124 in
2003.
Despite much early success, proteomic studies face
several challenging obstacles. Proteins are extremely di-
verse with large variations in size, charge, and solubility.
The extreme variation in protein concentration—thought
to vary over a range as high as 1010 (from serum albumin
at 40 mg/mL to cytokines in picograms/mL)— causes
enormous problems in designing devices that can detect
proteins of low abundance in the milieu of more abundant
proteins. Alternative splicing coupled with posttransla-
tional modifications significantly increases the number of
protein isoforms. Furthermore, no simple amplification
procedure such as polymerase chain reaction exists for
proteins. These obstacles, combined with the lack of a
defined endpoint, make proteomic studies quite intimidat-
ing yet exciting in that the real goal is to define how all
proteins come together to allow living organisms to func-
tion.
PROTEOMIC TECHNIQUES
The field of proteomics is driven by technology, which
allows for the systematic and rapid analysis of a large num-
ber of proteins. The trend is toward technologies that em-
phasize increased throughput and sensitivity. The heteroge-
neous nature of proteins necessitates the development of a
variety of methodologies to fully characterize their functions.
No single method exists that provides a detailed enough
snapshot for understanding all of a protein’s properties from
its size, sequence, three-dimensional structure, posttransla-
tional modifications, relative amounts, or enzymatic activi-
ties. In general, proteomic studies have often begun with one
of a variety of separation modalities such as gel electro-
phoresis or high performance liquid chromatography
(HPLC) to divide complex biologic samples into more man-
ageable fractions before analysis in a mass spectrometer.
Separation Technologies
Gel electrophoresis.—An indispensable technique in
molecular biology that set the stage for the large-scale
characterization of the proteome is gel electrophoresis.
The first gel-based forms of electrophoresis were devel-
oped during the late 1950s and have evolved significantly
during the past 50 years. The technique remains a staple
of modern labs— used to isolate and purify proteins, ana-
lyze the constituents of complex mixtures, and as a pre-
parative tool.
Gel electrophoresis separates proteins based on differ-
ences in their physical characteristics such as size and
charge (isoelectric point). Proteins migrate through a gel
matrix (usually acrylamide) in response to an electric
field. The most common method for separating proteins is
based on size. A detergent, sodium dodecyl sulfate (SDS),
is added to denature and coat proteins with a strong nega-
tive charge. SDS binds to most proteins in approximately
the same amount per mass of protein, resulting in their
migration through a gel based on size: smaller proteins
migrate more rapidly than larger proteins. A second tech-
nique, isoelectric focusing, separates proteins according to
their isoelectric points under denaturing conditions such
as urea. A third method separates proteins under nondena-
turing or native conditions based on intrinsic charge.
After gel electrophoresis, proteins can be identified or
visualized using a variety of methods. The simplest
method is staining, most commonly with either Coomas-
sie blue stain or the more sensitive silver stain. Each stain
allows for the visualization of proteins in a gel; Coomas-
sie to the mid-picomole (10⫺12) and silver stain to the
mid-femtomole (10⫺15) range. A protein’s mass is approx-
imated by comparing its length of gel migration with that
of known molecular weight standards. Protein abundance
is estimated by the intensity of gel staining.
A more informative technique for identifying specific
proteins in a gel is by Western blotting. Similar to South-
ern and Northern blotting, which probe for DNA and
RNA, respectively, Western blotting is a method to iden-
tify proteins in a gel using antibodies of known specific-
ity. After gel electrophoresis, a nitrocellulose membrane
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 AZOK ET AL
Figure 1. Two-dimensional gel electrophoresis. A sample of hu-
man plasma separated on a two-dimensional gel. The sample was
first loaded onto the gel and proteins were separated in the first di-
mension by pI (isoelectric point). Next, proteins were separated in
the second dimension by size. After electrophoresis, the gel was
stained and proteins were visualized as darkened spots. The com-
plexity of the plasma proteome is evident by the large number of
resolved spots. (Reproduced with permission from: Swiss Institute of
Bioinformatics, Geneva, Switzerland. Available at: http://www.
expasy.org/cgi-bin/map2/noid?PLASMA_HUMAN) copyright ©
Swiss Institute of Bioinformatics, Geneva, Switzerland.
is placed (or blotted) against the gel, allowing proteins to
diffuse out of the gel and adhere to the nitrocellulose
membrane. Through the sequential use of a primary anti-
body, secondary antibody, and enzyme substrate, a pro-
tein can be reliably identified in a complex mixture with
high sensitivity. Western blotting is commonly used to
corroborate genomic experimental data.
A third method to identify proteins from a gel is to
excise the gel fragment containing the region of interest,
elute the proteins from the gel, and subject the peptides/
proteins to further analysis such as mass spectrometry
(discussed in the following section).
A significant advance came in the early 1970s with the
description by O’Farrell and Klose of a method to com-
bine two electrophoretic techniques in orthogonal direc-
tions (2,3). Referred to as two-dimensional polyacryl-
amide gel electrophoresis (2D-PAGE), it substantially
increased the resolving power of gels from 100 proteins
on a conventional one-dimensional (1D) gel to approxi-
mately 1,000 to 2,000 proteins on a two-dimensional (2D)
gel (Fig. 1). The availability of 2D gels made it possible
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Reprinted from Academic Radiology, Vol 11, No 8, August 2004
to study the protein content of complex biologic samples,
such as whole blood or cell extracts.
Two-dimensional gel electrophoresis remains as the
most powerful method available to resolve a complex
protein mixture. The use of 2D gels for proteomic profil-
ing allows for the systematic and quantitative analysis of
a large number of proteins simultaneously. Typically, the
pattern of proteins seen in one biologic state is compared
with another, and any variation in protein expression can
be identified and studied further. One of the most suc-
cessful early protein profiling studies led to the discovery
of the tumor suppressor protein, p53 (4). On a 1D protein
gel, it was found that p53 was overexpressed when nor-
mal cells were compared with cells infected with a simian
virus 40 (SV40) DNA tumor virus. Two-dimensional gels
have been used extensively to characterize the proteins in
complex biologic samples such as tissue lysates and hu-
man blood.
Many refinements in 2D gel technology have been
made in the past 30 years. Improvements in gel reproduc-
ibility, resolution, bioinformatics, and mass spectrometry
technology have all improved its capability. The develop-
ment of immobilized pH gradients, in which the pH gra-
dient is fixed within the matrix, made experiments much
more reproducible. The introduction of narrow immobi-
lized pH gradients (zoom gels) significantly increased the
resolution. Furthermore, the development of differential
in-gel electrophoresis greatly improved the capability of
quantifying proteins in a gel.
However, 2D gel experiments remain very time-con-
suming, cumbersome, and are plagued by reproducibility
issues. A significant amount of sample is required for
each experiment, which often precludes its use in clinical
studies in which little sample is available. Analyzing the
hundreds to thousands of proteins resolved on a gel is
time-consuming even with improved commercial soft-
ware. The dynamic range achieved by current 2D gel
staining techniques is only three to four orders of magni-
tude. In comparison, the dynamic range of protein con-
centration in a cell can span 8 or even 10 orders of mag-
nitude in the blood. Furthermore, gel electrophoresis is
inherently a descriptive technique; any upregulation or
downregulation of a protein must be investigated by fur-
ther experiments.
HPLC.—A second major technique used to separate
biologic samples is HPLC. This technique separates com-
plex mixtures with high resolution based on a protein’s
physical and chemical properties. Advances in packing
materials coupled with the development of smaller col-
 Reprinted from Academic Radiology, Vol 11, No 8, August 2004 PROTEOMICS: THE LARGE-SCALE STUDY OF PROTEINS
Figure 2. High performance liquid chromatography (HPLC) chro-
matogram of proteins from Caenorhabditis elegans. Chromato-
gram of proteins from C. elegans separated via HPLC. Each of
the labeled peaks represents a unique protein. Peaks: 1, ribo-
somal protein S3A; 2, calreticulin precursor; 3, ribosomal protein
L1; 4, elongation factor 1-alpha; 5, malate dehydrogenase; 6, 40S
ribosomal protein; 7, vitellogenin; 8, arginine kinase; 9, HSP-1
heat shock 70-kDa protein A; and 10, ribosomal protein L7Ae.
The high resolving power of HPLC makes it an effective tool in
proteomic experiments. (Reproduced with permission from: Anal
Chem 2004;76:728 –735; ACS publications, copyright American
Chemical Society.)
umn diameters and high-pressure systems have increased
the resolution and capability of this procedure tremen-
dously. Today, it is widely used by protein chemists for
the purification of proteins and peptides prior to analysis
in a mass spectrometer.
Chromatography separates complex mixtures by virtue
of a stationary and a liquid phase. The development of
high-pressure systems in the late 1970s led to the devel-
opment of HPLC, which used high pressure to force the
mobile phase through the stationary phase at a high rate,
resulting in more rapid separation with enhanced resolu-
tion. Separation is dependent on a protein’s ability to ad-
here or diffuse through the stationary phase. Proteins are
effectively separated via HPLC by the heterogeneity
found within their amino acid sequence (primary struc-
ture) three-dimensional structure (secondary, tertiary, and
quaternary structure), and physical properties such as size
and isoelectric point (Fig. 2).
There is a wide variety of HPLC separation techniques
available depending on the choice of the stationary and
mobile phases. Among the most widely used techniques
are ion-exchange, size-exclusion, and reversed-phase,
which separate proteins by their charge, size, and hydro-
phobicity respectively. Methods to elute the sample from
the various columns are typically achieved by varying the
pH, salt concentration, or hydrophobicity of the mobile
phase. Proper choice of the optimal conditions combined
with the sequential use of two columns can result in a
nearly homogenous sample. Advances in HPLC technol-
ogy combined with its linkage to mass spectrometry (dis-
cussed in the following section) have transformed this
technology from its utilization mainly by chemists to
widespread use in biologic laboratories.
Mass Spectrometry
Mass spectrometry is a technique for determining the
molecular mass of intact molecules such as peptides or
proteins. It has evolved considerably in the past 15 years
and has only become suitable for proteomic experiments
with the availability of genome sequence databases. To-
day, it is the most sensitive and rapid method for identi-
fying and quantifying proteins from complex samples.
The sensitivities of high-end instruments can detect pro-
teins in the femtomolar (10⫺15) to attomolar (10⫺18)
range. The high degree of mass accuracy (better than
0.01%) and resolution of modern mass spectrometers can
accurately detect posttranslational modifications such as
phosphorylation or glycosylation. The data from an exper-
iment are a spectrum containing a mass/charge (m/z) ratio
for each protein/peptide along with its intensity. Mass
spectrometry experiments are most often preceded by a
technique (usually gel electrophoresis or HPLC) to sim-
plify the biologic sample before analysis.
Mass spectrometers consist of an ion source, mass ana-
lyzer, and a detector. The procedure relies on sample ioniza-
tion and transfer into the gas phase in a vacuum chamber.
The ion source ionizes and desorbs the sample into the gas
phase to facilitate its analysis. A key breakthrough in the
history of mass spectrometry was the discovery during the
late 1980s of two “soft” methods to efficiently transfer pro-
teins into the gas phase without breaking them apart: matrix-
assisted laser desorption/ionization (MALDI) and electro-
spray ionization (ESI). In MALDI experiments, an ultravio-
let laser is directed at the protein sample, resulting in the
simultaneous ionization and desorption of the protein into
the gas phase. In ESI experiments, the sample is sprayed
through a microcapillary tube and into the vacuum chamber
across a potential difference. After sample introduction into
the mass spectrometer, a mass analyzer separates ions via
differences in their mass to charge ratios before their colli-
sion with the detector. A wide variety of mass analyzers is
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 AZOK ET AL Reprinted from Academic Radiology, Vol 11, No 8, August 2004

Figure 3. Tryptic digestion and identification of a protein via mass spectrometry. (a)
Matrix-assisted laser desorption/ionization mass spectrum of a tryptic digestion of an
RNA polymerase I–specific transcription initiation factor. Each peak represents a frag-
ment of the parent ion generated through digestion of the protein with trypsin. (b) Pep-
tide fragments of the RNA polymerase I-specific transcription initiation factor detected
in the mass spectrum. An unknown protein is identified by entering the mass of the
fragment ions into a specialized protein database. (Adapted with permission from: Anal
Chem 2004;76:2196 –2202, ACS publications, copyright American Chemical Society.)

available such as time-of-flight (TOF), quadrupole, and ion
trap.
Two main methods exist for identifying proteins in a
mass spectrometer: peptide-mass mapping and collision
induced decay (CID) using tandem mass spectrometry
(MS/MS). Peptide mass mapping is preceded by diges-
tion of the protein sample with an enzyme of known
cleavage specificity (typically trypsin). Next, the di-
gested sample is analyzed in a MALDI-TOF experi-
ment. The pattern of observed MS peak values, repre-
senting the protein fragments of the parent ion, is com-
pared with the predicted protein fragments of all the
proteins within a database (Fig. 3). Based on the mass
to charge ratios of peptide fragments detected com-
pared with those predicted, a protein can be identified
with a high degree of accuracy. CID using MS/MS is a
second technique used to characterize unknown pro-
teins. It consists of two mass analyzers (typically an
ion trap or triple-quadrupole); the first isolates a partic-
ular peptide/protein of interest by its m/z ratio, whereas
the second fragments the parent ion into many second-
ary fragment ions. The mass difference between ions is
the molecular weight of individual or groups of amino
acids. This technique has the added advantage com-

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 Reprinted from Academic Radiology, Vol 11, No 8, August 2004 PROTEOMICS: THE LARGE-SCALE STUDY OF PROTEINS
pared to peptide mass mapping of providing informa-
tion about a protein’s amino acid sequence.
Advances in mass spectrometry combined with 2D-gel
technology has made it routine to excise a protein of in-
terest from a gel, elute the protein, and identify it via a
mass spectrometry experiment (Fig. 4). Today, this proce-
dure is widely used to catalog proteins found within dif-
ferent biologic samples and to study proteins up or down-
regulated in various physiologic and pathologic states.
Liquid chromatography has been coupled to MS/MS
(LC-MS/MS) to provide even greater resolution of the
proteins present in complex sample mixtures. The advan-
tage of coupling HPLC to a mass spectrometer is that the
process can be automated to accommodate high-through-
put experiments analyzing hundreds, even thousands, of
proteins. The potential of these technologies was shown
in a recent study of the Plasmodium falciparum life cycle,
using multidimensional protein identification technology,
which combines HPLC and MS/MS (5). A total of 2,415
proteins expressed in one of the four parasite life cycles
was identified. These proteins represented 46% of the
5,276 gene products encoded by the Plasmodium genome.
In particular, stage-specific proteins were identified that
allow for the identification of stage-specific molecular
events. Furthermore, more than 100 proteins were identi-
fied that have little or no homology to human proteins.
These proteins have great potential as Plasmodium-spe-
cific targets for new drugs and vaccines.
Structural Genomics
The field of science that investigates the three-dimen-
sional structure of proteins is structural genomics. Similar
to proteomic projects, structural genomic studies rely
heavily on high-throughput technologies to efficiently
characterize the three-dimensional structure of a large
group of proteins at high resolution. Structural biologic
data for individual proteins is absolutely crucial for un-
derstanding biological processes such as the mechanisms
of various drugs, signal transduction pathways, and prop-
erties of cell surface receptors. This information can be
used in the development of novel drugs and for under-
standing how a genetic mutation affects the properties of
a particular protein. A major goal of structural genomics
is to characterize all protein folds to facilitate the three-
dimensional determination of entire proteomes.
The major techniques used by structural biologists are
x-ray crystallography, nuclear magnetic resonance
(NMR), and electron microscopy. These three methods
can all provide high-resolution structural data of individ-
ual proteins and large macromolecular protein complexes
such as the ribosome and cell membranes. All three meth-
ods give slightly different, but often complementary,
structural data that can be used in parallel. Although
much has been learned about the general rules that govern
protein folding from thousands of high-resolution three-
dimensional protein structures, there is still no method to
accurately predict how a protein will fold without deter-
mining it experimentally. The largest repository of protein
structural data, the Protein Data Bank, contains 22,936
protein structures (as of May 8, 2004) (6).
The most widely used method for analyzing protein
structures is x-ray crystallography. It provides the high-
est structural resolution of all three techniques and re-
quires the synthesis of protein crystals from large quanti-
ties of purified protein. Protein crystals are subsequently
subjected to an x-ray beam whose diffraction pattern is
captured and converted to an electron density map. The
known protein amino acid sequence is fit to the electron
density map and a three-dimensional model of the protein
is generated (Fig. 5). Much of the process has been auto-
mated to facilitate high-throughput studies.
Similar to x-ray crystallography, nuclear magnetic
resonance provides protein structural data at the atomic
level. A major limitation of NMR is its inability to ana-
lyze proteins larger than 20 kDa. However, NMR does
not require protein crystals, and is therefore applicable for
the structural determination of proteins to which crystals
are difficult to generate, such as transmembrane proteins.
Furthermore, NMR can be used to study protein-ligand
interactions and dynamic processes, such as protein stabil-
ity and enzyme catalysis. NMR experiments are con-
ducted in the presence of a strong magnetic field and rely
upon the ability of nuclei (such as 1H and 13C) to resonate
in response to a radiofrequency pulse. The exquisite reli-
ance of nuclear resonance on its electronic environment
forms the basis for the use of NMR in protein structure
determinations.
Electron microscopy, as with x-ray crystallography
and NMR, is used to provide structural data of biologic
samples, but at much lower resolution. The advantage of
this technique is that smaller quantities of material are
required and it is not as dependent on protein purity.
Electron microscopy is becoming increasingly popular for
the study of large macromolecular complexes such as vi-
ruses and ribosomes. A range of magnifications is avail-
able to image a wide variety of molecular structures.
The data generated by each of the three aforemen-
tioned techniques are used synergistically to aid in the
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Figure 4. Studying a proteome by two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization (MALDI)-
time of flight (TOF). (a) The sample was first separated on a two-dimensional gel; the portion of the gel containing the protein of interest
was excised for mass spectrometry analysis. (b) Analysis of the protein sample on a MALDI TOF mass spectrometer. (c) The protein was
definitively identified through detection of the tryptic protein fragments and the use of specialized protein databases. (Reproduced with
permission from: Brown TA. Genomes. 2nd ed. [Figure 72.4] Oxford, UK: BIOS Scientific Publishers Ltd; 2002.)

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 Reprinted from Academic Radiology, Vol 11, No 8, August 2004 PROTEOMICS: THE LARGE-SCALE STUDY OF PROTEINS
Figure 5. X-ray crystallography. (a) Protein crystal of ribulose
bisphosphate carboxylase. (b) X-ray diffraction pattern obtained
from the crystal. (c) Simplified model of the protein structure de-
rived from the X-ray diffraction data. (Reproduced with permission
from: Alberts B. et al. Molecular biology of the cell. 3rd ed. New
York: Garland Publishing; 1994.)
understanding of biologic processes. This is best illus-
trated by the combined use of all three technologies in the
structural determination of the ribosome. Early studies
used low-resolution electron microscopy experiments and
a combination of NMR and x-ray crystallography to char-
acterize small ribosomal components. Finally, an essen-
tially complete ribosome complex was crystallized, lead-
ing to a high-resolution ribosome crystal structure in
which individual atoms can be visualized. This has led to
a more comprehensive understanding of protein synthesis
and new insight into how various antibiotics interact with
the ribosome at the atomic level.
Functional Assays
Within cells, proteins interact to form complex multi-
subunit structures, which play important biologic roles.
An example is the many transcription factors, enhancers,
and repressor proteins that come together during tran-
scription to regulate the coding of a specific mRNA tran-
script from DNA. Understanding all of the possible inter-
actions that regulatory proteins make with other proteins
would be valuable in understanding the biology of the
system and open up new insights to how the cell is put
together. Several techniques and assays have been devel-
oped to better understand protein-protein interactions; two
are described in the following section.
The most widely used technique for identifying pro-
tein-protein interactions has been the yeast two-hybrid
system. The process involves fusion of a protein to a
DNA binding domain (the “bait”) and the concurrent fu-
sion of a second protein (the “target) to an activation do-
main of a transcriptional activator (Fig. 6). The interac-
tion of the two proteins results in transcription of a re-
porter gene, a process that can be measured. In a large-
scale yeast two-hybrid screen involving the yeast,
Saccharomyces cerevisiae, 957 interactions between 1,004
different yeast proteins were identified (7). In many cases,
proteins of previous unknown function were put into
some biologic context by their interaction with other
known proteins. Furthermore, biologic pathways, which
previously were not thought to be connected, were linked
through the detection of novel interactions between pro-
teins with different biologic functions.
A technique that has great potential for probing pro-
tein-protein interactions is protein microarrays. Similar to
DNA microarrays, protein microarrays consist of proteins
spotted directly onto a chip surface. A major challenge is
attaching proteins to the chip without denaturing them.
Several studies have demonstrated the feasibility of this
technique, both for maintaining the activity of affixed
proteins and for maintaining high-specificity interactions.
This was shown by the detection of a single specific in-
teraction on a protein array among 10,799 copies of a
second protein (8). Challenges ahead for this procedure
are the development of novel methods for attaching pro-
teins to the chips while maintaining biologic activity,
miniaturizing the technique to facilitate the study of large
numbers of proteins, and improved methods for purifying
the proteins needed to make these arrays.
BIOINFORMATICS
Proteomic experiments have necessitated improvements
and advances in bioinformatics to efficiently manage and
analyze the large amounts of data that are generated. For-
tuitously, many of the same techniques of data analysis
and data handling developed for genomic experiments can
be rapidly modified for use in proteomic experiments.
Advances in computer technology have greatly facilitated
the storage and analysis of large data sets leading to the
creation of large public databases. The concurrent devel-
opment of specialized software and increased variety of
statistical techniques has enabled the field of proteomics
to move forward.
A challenge posed by proteomic experiments is how to
best disseminate the acquired data. Publications in peer-re-
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 AZOK ET AL Reprinted from Academic Radiology, Vol 11, No 8, August 2004

Figure 6. Yeast two-hybrid system. Interaction of the “bait” protein and the “target”
protein bring together the GAL4 activation domain (AD) and binding domain (BD). The
function of the GAL4 protein is restored, resulting in transcription of the reporter gene,
which can be measured. (Reproduced with permission from: Griffiths AJ et al. Modern
genetic analysis. New York, NY: W.H. Freeman & Co; 1999.)

viewed journals provide a concise summary of the results,
but often lack important details and rarely provide access to
raw data. Many journals now offer links to supplementary
material on their respective web sites, and some laboratories
provide raw data on web sites for others to analyze. The
exchange of experimental data greatly improves cooperation
among different laboratories and allows for the independent
validation of experimental results. An example is the clinical
proteomics program at the National Institutes of Health
(http://ncifdaproteomics.com/), where various groups have
used different statistical techniques to evaluate the same raw
data. This has led to confirmation of the results and the abil-
ity to compare different techniques of analysis.
Many proteomic databases are available to researchers
worldwide through the World Wide Web. These data-
bases allow for the immediate availability of data and for
enhanced features compared with text-based journals.
Among the most widely used databases are SWISS-
PROT, TrEMBL (Translation of the EMBL [European
Molecular Biology Laboratory] Nucleotide Sequence Da-
tabase), 2D gel, and three-dimensional crystal structure
databases. The SWISS-PROT database, in particular, con-
tains a plethora of protein data including information
about protein function, posttranslational modifications,
structural data, and links to more than 50 other databases
(9). SWISS-PROT (as of May 8, 2004) has 149,914 en-
tries. As these databases have evolved, they have become
more complete with less redundancy and better integra-
tion.
CLINICAL PROTEOMICS
Proteomics, although poised to make significant contri-
butions to the understanding of disease pathogenesis,
promises to make significant contributions to clinical
medicine, even within the next 5 years— especially in the
areas of disease diagnosis and development of new drugs.
This has generated an enormous amount of excitement
and several companies have formed to address this issue.
A large amount of effort is currently focused on study-
ing proteins found in the blood of patients with a variety

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 Reprinted from Academic Radiology, Vol 11, No 8, August 2004 PROTEOMICS: THE LARGE-SCALE STUDY OF PROTEINS
of diseases. The hypothesis is that blood continually
bathes all tissues and that any physiologic or pathologic
process is represented by proteins and their fragments in
the blood. Although single-marker protein tests such as
prostate-specific antigen and carcinoembryonic antigen
have some clinical efficacy, their sensitivity and specific-
ity are far from perfect. Using several proteins in combi-
nation, so-called protein pattern diagnostics, has been
shown in several studies to give sensitivities and specific-
ities near 100%.
Many of the studies that have attempted to identify
protein biomarkers have relied on a technique called sur-
face-enhanced laser desorption/ionization mass spectrome-
try (SELDI-MS). Similar to gel electrophoresis,
SELDI-MS is used to study the expression patterns of a
large number of proteins simultaneously. At the core of
this technology are specially designed protein chips with
various chromatographic surfaces that bind and retain a
subset of proteins based on their chemical properties. The
advantage over traditional MALDI techniques is the rapid
characterization of protein populations in crude biologic
samples such as serum or tissue lysate. Furthermore, in
contrast to 2D gel electrophoresis, SELDI-MS is high-
throughput and relatively simple.
Many of the experiments using this technology have
focused on the identification of serum protein markers for
various neoplastic processes, especially ovarian cancer. A
landmark study in 2002 identified a protein pattern in the
blood that could segregate ovarian cancer patients from
normal controls with sensitivities and specificities near
100% (10). Other studies have shown characteristic pro-
tein patterns in the blood for prostate cancer and in the
cerebrospinal fluid for Alzheimer’s disease. This tech-
nique, in particular, has created a great deal of excitement
for the development of diagnostic tests based on protein
patterns in different biologic compartments.
The field of proteomics has the potential to accelerate
the discovery and development of new drugs. The vast
majority of drugs is directed against a small number of
protein targets, approximately 500. This number is likely
to grow significantly as proteomic experiments identify
new protein targets and as more high-resolution crystal
structures are obtained, which make rational drug design
possible. Furthermore, proteomic studies have proven use-
ful in elucidating the mechanisms of drug action and in
understanding the pharmacologic and toxic effects of
drugs. Most major pharmaceutical companies have imple-
mented proteomic programs and the trend toward using
proteomic technologies in all phases of drug development
is sure to continue as the technology improves.
APPLICATIONS FOR IMAGING
Proteomic technologies have great potential for making
significant contributions to the field of radiology. Whereas
most current imaging technologies such as magnetic reso-
nance imaging and computed tomography rely on identi-
fying anatomic relationships between different organs and
tissues, the future will most likely rely more on functional
and molecular or cellular specific imaging—so called mo-
lecular imaging. Proteomics will most likely have the
greatest impact in the identification of new targets to
which imaging probes and drugs can be designed to spe-
cifically target disease processes. Furthermore, the incor-
poration of knowledge from other fields such as chemistry
and nanotechnology will aid in the development of tar-
geted probes and therapies, which will greatly enhance
our ability to follow, treat, and assess the effectiveness of
clinical therapies.
Summary
Advances in protein separation techniques, mass spec-
trometry, and the availability of complete genome se-
quence databases have led to enormous growth of the
field of proteomics over the past decade. Together with
genomics, proteomics promises to provide a comprehen-
sive understanding of how the DNA sequence ultimately
leads to the synthesis of proteins and how these proteins
are influenced and affected by various physiologic and
pathologic processes. Hopefully, the knowledge provided
by genomic and proteomic experiments will be rapidly
translated into improved clinical therapies. In the future,
proteomics will assume a much larger role in biomedical
research as improvements in instrumentation and automa-
tion of existing technologies lead to increasingly valuable
and data-rich experiments.
Glossary
Two-dimensional polyacrylamide gel electrophoresis
(2D-PAGE)
A method for separating proteins in two dimensions on
a gel. Typically, proteins are separated in the first
dimension by pI (isoelectric point) and in the second
dimension by size. 2D-PAGE has much greater
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 AZOK ET AL
resolution than one-dimensional gels and is widely used
for analyzing complex protein mixtures.
Electron microscopy
A microscopic technique that uses a beam of electrons
to visualize extremely small objects. Electron
microscopes have much greater resolution than light
microscopes.
Gel electrophoresis
A gel-based method for separating a mixture of
molecules. A current is applied and negatively charged
molecules travel toward the positively charged cathode.
Sodium dodecyl sulfide (SDS) is used to coat proteins
with a uniformly negative charge so that proteins are
separated by size.
High performance liquid chromatography (HPLC)
A chromatographic technique using high pressure to
force the analyte (liquid phase) through a column (solid
phase). The high pressure significantly decreases the
amount of time the analyte remains on the stationary
phase and results in narrow peaks and, therefore, better
selectivity. A variety of solid and liquid phases is
available for purification of nearly any sample.
Mass spectrometry
A method for separating proteins based on their mass
to charge (m/z) ratios. A mass spectrometer consists of
an ion source, mass analyzer, and a detector. The ion
source ionizes and vaporizes the sample into the mass
spectrometer. The mass analyzer separates the ions and
the detector records the current of ions on their exit
from the mass analyzer.
Nuclear magnetic resonance (NMR)
A technique used to study the three-dimensional
structure of proteins that relies on the resonance of
protons to radiation within a strong magnetic field.
X-ray crystallography
A technique for characterizing the three-dimensional
structure of proteins. The sample is first crystallized
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Reprinted from Academic Radiology, Vol 11, No 8, August 2004
and subsequently bombarded by x-rays. The x-ray
diffraction pattern is recorded and analyzed to produce
an electron density map to which the known amino
acid sequence is fit. X-ray crystallography can provide
extremely high-resolution structures, to single
Angstroms.
REFERENCES
1. Human Proteome Organisation. HUPO mission statement. Available at:
http://www.hupo.org/mission.htm. Accessed May 8, 2004.
2. O’Farrell PH. High resolution two-dimensional electrophoresis of pro-
teins. J Biol Chem 1975;250:4007– 4021.
3. Klose J. Protein mapping by combined isoelectric focusing and electro-
phoresis of mouse tissues. A novel approach to testing for induced
point mutations in mammals. Humangenetik 1975;26:231–243.
4. Crawford LV, Pim DC, Gurney EG, Goodfellow P, Taylor-Papadimitriou
J. Detection of a common feature in several human tumor cell lines—a
53,000-dalton protein. Proc Natl Acad Sci 1981;78:41– 45.
5. Florens L, Washburn MP, Raine JD, et al. A proteomic view of the
Plasmodium falciparum life cycle. Nature 2002;419:520 –526.
6. Research Collaboratory for Structural Bioinformatics (RCSB) Protein
Data Bank (PDB). Available at: http://www.rcsb.org/pdb. Accessed May
8, 2004.
7. Uetz P, Giot L, Cagney G, et al. A comprehensive analysis of protein-
protein interactions in Saccharomyces cerevisiae. Nature
2000;403:623– 627.
8. MacBeath G, Schreiber SL. Printing proteins as microarrays for high-
throughput function determination. Science 2000;289:1760 –1763.
9. ExPASy (Expert Protein Analysis System) proteomics server of the
Swiss Institute of Bioinformatics. Available at: http://www.expasy.org.
Accessed May 8, 2004
10. Petricoin EF, Ardekani AM, Hitt BA, et al. Use of proteomic patterns in
serum to identify ovarian cancer. Lancet 2002;359:572–577.
FURTHER READING
Additional Reference Material
Conn PM. Handbook of proteomic methods. Totowa, NJ: Humana Press;
2003.
Hanash S. Disease proteomics. Nature 2003; 422:226 –232.
Pennington SR, Dunn MJ, Proteomics from protein sequence to function.
New York: BIOS Scientific Publishers; 2001.
Tyers M, Mann M. From genomics to proteomics. Nature 2003; 422:193–
197.
Westermeier R, Naven T. Proteomics in practice. Weinheim, Germany:
Wiley-VCH; 2002.

A Primer on Molecular Biology for Imagers:
S. Narasimhan Danthi, PhD, Sunil D. Pandit, PhD, and King C.P. Li, MD, MBA
Imagine administering a chemical in a live animal, look-
ing at its interactions at molecular and cellular level, and
identifying in real time various physiologic and patho-
logic conditions. To many people, this idea may feel like
science fiction. Novel chemicals that are in research and
development should help to make such a scenario a re-
search and clinical reality in many applications. These
chemicals under investigation that are used to probe mo-
lecular function at the molecular and cellular level in
vitro, ex vivo, and or in vivo are called molecular probes
(MPs). They provide high sensitivity for imaging minute
molecular systems such as receptors, enzymes, and trans-
porters. MPs are classified according to the imaging tech-
niques with which they are used, and the selection of
MPs is dependent on the type of imaging modality. MPs
can be radioactive or nonradioactive and be designed to
image endogenous or exogenous genes, messenger ribo-
nucleic acid transcripts, or the expressed proteins (recep-
tors, enzymes, and transporters). MPs that target proteins
have been a major focus because they are expressed in
large numbers (thousands to millions of copies per cell)
and can allow the accumulation of the MPs in a specific
site or tissue and thus can produce good signal-to-noise
ratio. Also, protein expression is the ultimate result of
genetic processing and hence imaging protein expression
is a good choice.
Imaging is mainly used for assessing morphology non-
invasively. However, much excitement has been generated
regarding the potential of using MPs to image specific
Reprinted with permission from Acad Radiol 2004; 11:1047–1054
1 From the Molecular Imaging Laboratory, Department of Diagnostic Radiol-
ogy, National Institutes of Health, Clinical Center, Building 10, 9000 Rock-
ville Pike, 1N306, Bethesda, MD 20892 (S.N.D., S.D.P., K.C.P.L.). Received
June 18, 2004; accepted June 19, 2004. Address correspondence to
K.C.P.L. e-mail: kingli@nih.gov
© AUR, 2004
doi:10.1016/j.acra.2004.10.008
Chapter VII
VII. Molecular Imaging Probes1
events at the molecular and cellular level. Molecular im-
aging can potentially be used to study biology, diagnose
pathologic conditions, guide therapy, and monitor re-
sponses. There are several methods of classifying MPs.
Based on their specificity, they can be classified into spe-
cific probes (targeted probes) and nonspecific (untargeted
probes). In targeted probes, the target of interest is ex-
pressed selectively in abundance and the MPs should se-
lectively bind to the target with high affinity. The MPs
should be cleared rapidly from nontarget tissues and the
MPs should be stable in vitro and in vivo. This review
will focus on MPs based on their use in various imaging
techniques.
MOLECULAR PROBES FOR NUCLEAR
MEDICINE IMAGING
Positron emission tomography (PET) and single photon
emission computed tomography (SPECT) are the two im-
portant techniques used in Nuclear Medicine Imaging.
Both PET and SPECT require radioactive probes. Radio-
isotope is incorporated into the probe either covalently
(18F, 11C) or attached noncovalently (111In) using chelators
such as diethylenetriaminepentaacetate. One of the first
used PET MP is 2-[18F] fluoro-2-deoxy-D-glucose (FDG).
Clinically, FDG is still widely used as a PET MP. Struc-
turally, FDG resembles D-glucose (Fig. 1) and thus enters
the cells using glucose transporter type 1 (Glut1). Wher-
ever there is an increased glucose uptake, FDG is also
taken up increasingly. Subsequent to its uptake, similar to
glucose, FDG is phosphorylated by hexokinase (Fig. 2).
However, unlike glucose-6-phosphate, FDG-6-phosphate
is not further metabolized and gets trapped in the cells;
therefore, the trapped radiotracer can be imaged. This MP
can be used to image increased glycolytic activity (as
seen in tumors), increased hexokinase activity, and up-
regulated Glut1. FDG thus tracks the glucose metabolism,
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 DANTHI ET AL
Figure 1. Structural comparison between fluoro-2-deoxy-D-glu-
cose and glucose.
Figure 2. Mechanism of fluoro-2-deoxy-D-glucose accumula-
tion.
hexokinase activity, and Glut1 transporter and is taken up
into all glucose-using cells, such as macrophages. As a
result, FDG is not selective for tumor; imagers need to
consider this when interpreting the imaging data. Today
PET imaging with FDG is a widely used method for im-
aging normal and pathologic functions, especially tumors
of brain, heart, and other tissues (1).
Choline (Fig. 3) uptake and its phosphorylation by
choline kinase are known to be increased in tumor cells.
PET MPs, including [11C]choline, [18F]fluoroethylcholine,
and [18F]fluoromethylcholine, were developed to image
tumors of the brain, lung, head and neck, colon, bladder,
and primary and metastatic prostate. In clinical imaging
studies of prostate cancer, [18F]fluoromethylcholine has
been shown to be more accurate than FDG. Somatostatin
receptors are known to be expressed on tumor cells of
neuroendocrine (pituitary and pancreas) as well as non-
neuroendocrine origin (lymphomas and breast) (2– 4). Oc-
treotide binds to somatostatin, and octreotide-containing
MPs have been developed that can image the expressed
somatostatin receptors. DOTA-octreotide and TETA-oct-
reotide (Fig. 4) have been developed with radionuclides
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Reprinted from Academic Radiology, Vol 11, No 9, September 2004
Figure 3. Structure of choline.
Figure 4. Structure of DOTA and TETA octreotide.
such as 111In, 99mTc, 68Ga, 18F, 86Y, and 64Cu for nuclear
medicine imaging.
Integrin ␣v␤3 is known to be upregulated in angiogene-
sis and tissue remodeling of various diseases including
osteoporosis, rheumatoid arthritis, macular degeneration,
and cancer. Various groups have developed MPs targeting
integrins. Cyclic Arginine-Glycine-Aspartate (RGD) pep-
tide labeled with 125I showed high tumor uptake in
SPECT imaging. Neurotensin, a putative neurotransmitter
in the central nervous system, has been found to be ex-
pressed in pancreatic tumors. MPs with 111In and 99mTc
have been used to image neurotensin receptors, specifi-
cally neurotensin receptor subtype1. Several other pep-
tide- and peptidomimetic-conjugated MPs have been de-
veloped for PET and SPECT imaging of sodium/iodide
 Reprinted from Academic Radiology, Vol 11, No 9, September 2004
Figure 5. General structures of molecular probes for positron emission tomography
and single photon emission computed tomography.
symporter, multidrug resistance p-glycoprotein, and dopa-
mine receptor type 2. In PET MPs, the radionuclide is
attached covalently to the targeting agent, whereas in
SPECT the MPs are attached with a chelator, which can
chelate the radionuclide (Fig. 5).
IMAGING TRANSGENE USING PET/SPECT
We have discussed previously the herpes simplex virus
thymidine kinase gene (HSV1-tk) reporter– gene probe
system used for imaging transgene expression (5). The
other reporter– gene probe system commonly used for
PET imaging is the dopamine D2 receptor. The primary
objective of PET reporter genes is to obtain sufficient
signal from cells expressing the reporter gene from the
accumulation of the appropriate tracer. This enables imag-
ing to monitor the temporal changes in the magnitude and
location of the gene expression. The expression of the
reporter genes can be driven by tissue-specific promoter
or a generic strong promoter such as the cytomegalovirus
promoter. These reporter genes need to be delivered to
the site of imaging and have to express the protein before
the probe that they modify is injected for imaging. Viral
or liposomal delivery vehicles have been used for targeted
delivery, but mostly these genes have been used in xeno-
graft models in which the tumor-forming cells have been
previously transfected with the reporter gene.
Several different probes (substrates) have been devel-
oped for the HSV1-tk gene, which include the F-18 la-
beled analog of ganciclovir (a drug used to treat HSV),
F-18-penciclovir, and I-131 and I-124 labeled derivatives
of thymidine such as 2=-fluoro-2=-deoxy-1-␤-D-arabino-
furanosyl-5-iodo-uracil (6). The probe, when injected in-
travenously in the mouse, diffuses freely in and out of the
cell if there is no thymidine kinase activity. If HSV1-tk
gene is being expressed, the substrate gets phosphorylated
and trapped inside the cell. Because one molecule of the
kinase enzyme can phosphorylate many molecules of the
MOLECULAR IMAGING PROBES
substrate, this leads to amplification in the signal. Addi-
tionally, a mutant HSV1-tk gene has been developed
(HSV1-sr39tk) that is more sensitive to acycloguanosines
and is less sensitive to changes in intracellular concentra-
tions of thymidine.
In the dopamine D2 system, the dopamine D2 is the
receptor and the ligand used is [F-18] fluoroethylspipep-
rone. The dopamine D2 receptor is expressed on the cell
surface, and one molecule of the [F-18] fluoroethyl-
spipeprone ligand binds to a molecule of the receptor.
There is no amplification of the signal in this system, and
no significant dissociation of the ligand from the receptor
occurs during the experiment. Other PET reporter probe
systems that have been used include Na/I symporter and
the somatostatin receptor. Other reporter gene constructs
that are available include fusion of the HSV1-tk gene to
the fluorescent reporter green fluorescent protein and bidi-
rectional inducible therapeutic and reporter gene expres-
sion in which the therapeutic gene expression can be
monitored by imaging reporter gene expression.
MPs FOR MAGNETIC RESONANCE
IMAGING
There are two major classes of MPs for magnetic reso-
nance imaging (MRI). The first is of paramagnetic MPs
that are gadolinium (Gd)-based that generate T1 positive
signal enhancement; the second is of superparamagnetic
MPs that use iron oxide to generate strong T2-negative
contrast in MRI (Fig. 6). The Gd-based contrast agents
can be classified into small molecular and macromolecu-
lar MPs. There are several small molecular Gd contrast
agents that are commercially available (eg, Magnevist,
AngioMARK, OptiMARK) to image by magnetic reso-
nance. However, these agents are not targeted and hence
are nonspecific. Several macromolecular carriers have
been designed and tested and some of them are targeted
MPs. Protein-based macromolecular MPs (albumin Gd
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 DANTHI ET AL
Figure 6. Molecular probes for magnetic resonance imaging.
conjugates), monoclonal antibody– based MPs, poly-
amidoamine dendrimers of various generations with Gd
MPs (7), and nanoparticle-based MPs have been synthe-
sized and used in imaging with MRI. Chelated metal cat-
ions such as Gd, dysprosium, or superparamagnetic nano-
particles have been used as targeted or smart probes for
MRI. Polymerized nanoparticles with chelated Gd that are
targeted to angiogenic vessels in a rabbit VX2 carcinoma
model have shown promise in MRI (8).
The iron oxide– based T2 agents can be either monoc-
rystalline or polycrystalline iron oxide coated with poly-
saccharides, such as dextran. A number of dextran-coated
iron oxide–targeted particles have been developed for
␣v␤3 integrins, vascular cell adhesion molecule-1
(VCAM-1), E-selectin, and transferrin receptor. Iron ox-
ide particles with monoclonal antibodies targeted to leu-
kocytes have been developed for use with MRI. E-selec-
tin monoclonal antibody has been conjugated to iron ox-
ide particles, and these MPs have been used for in vitro
studies. Iron oxide–targeted MPs for imaging inflamma-
tion have been prepared by conjugating human polyclonal
immunoglobulin G (9). Imaging apoptosis by using iron
oxide conjugated to synaptotagmin has shown some
promise.
Smart, activatable agents have been developed for
MRI. In one enzyme activatable design, high-affinity
chelators were attached to Gd, preventing access to water
molecules, and the MRI baseline signal was suppressed.
This agent could be cleaved by the enzyme ␤-galactosi-
dase (product of lacZ gene), which restores full access of
water molecules to Gd, and the T1 relaxivity could be
measured (10). The other chemical principle used to de-
sign smart MRI contrast agents is the assembly and disas-
sembly of magnetic nanoparticles. Magnetic nanosensors
composed of magnetic nanoparticles can be used to detect
molecular interactions by MRI (11). Dextran-coated bio-
compatible monocrystalline iron oxide nanoparticles have
been designed to detect various kinds of biologic interac-
tions. When these magnetic nanosensors cluster together,
there is a decrease in spin-spin relaxation time (T2) of
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Reprinted from Academic Radiology, Vol 11, No 9, September 2004
surrounding water molecules, which can be measured by
MRI/nuclear magnetic resonance techniques. This concept
has been used to develop magnetic nanosensors that de-
tect DNA, proteins, viruses, and enzymatic activity such
as restriction endonuclease and methylation. Recent de-
signs of the smart magnetic nanoparticles include analysis
of telomerase activity and the measurement of myeloper-
oxidase activity, an enzyme shown recently to be elevated
in inflammation and coronary disease.
Recently, there has been interest in stem cells and their
potential applications in therapy. Researchers are inter-
ested in tracking the fate of these cells and determining
their percentage and location following injection in vivo.
CL-cross linked, IO-iron oxide particles attached with
human immunodeficiency virus-tat peptides have been
used to label hematopoietic and neural progenitor cells
and to track their fate in vivo using MRI (12). Other re-
searchers have used ferumoxide-labeled stem cells and
progenitor cells to track their fate in vivo using MRI.
MOLECULAR PROBES FOR ULTRASOUND
IMAGING
About 30 years ago, it was discovered that air bubbles
could be detected in the bloodstream after injections of
agitated aqueous solutions. Since then, considerable
progress has been made in the development of MPs for
ultrasound (US) imaging. MPs for US are made from bio-
degradable polymers such as lipids or proteins in which is
inserted a low solubility gas that is nonreactive, such as
perfluorocarbon (Fig. 7). As with MPs that are used for
other imaging modalities, the US MPs can also be classi-
fied into targeted (specific) and nontargeted MPs. The
clinically used microbubbles are usually very close in size
to those of the red blood cells and the rheologic proper-
ties of these MPs are also similar to that of red blood
cells. Thus these MPs can be used to map the vasculature
and determine blood flow, perfusion into, and the blood
volume of various tissues.
 Reprinted from Academic Radiology, Vol 11, No 9, September 2004
Figure 7. Molecular probes for ultrasound imaging.
The nontargeted MPs for US can be divided into three
types based on their design. The first type is an emulsion
of a gaslike perfluorocarbon with a surfactant that stabi-
lizes the gas. The second type is dry powder or a solution
in a vial with perfluorocarbon gas already filled in it. Be-
fore use, the contents of the vial are reconstituted, during
which a shell-covered microbubble dispersion is formed.
The third type of MP is made by mixing perfluorocarbon
gas with a medium that will quickly form a shell by self-
assembly using sonication; no further processing is re-
quired before use in patients or animals. Targeted MPs
for US imaging are under active research. Liposomes of
varying sizes with various antibodies covalently attached
to their surfaces have been studied with some promising
preliminary results.
MOLECULAR PROBES FOR OPTICAL
IMAGING
Optical imaging techniques include optical coherence
tomography, fluorescence imaging, and bioluminescence
imaging. We have discussed fluorescent and biolumines-
cence reporter genes and vectors previously (5). There are
many examples in the literature in which these reporter
genes have been applied as imaging agents for cell label-
ing and gene expression analysis.
Development of MPs for fluorescence imaging has
been a hot area of research. Several groups have devel-
oped targeted fluorescent probes that emit in the near-
infrared region (NIR) for in vivo applications. NIR light
(650 –900 nm) enables imaging at a greater depth because
hemoglobin, water, and lipids have their lowest absorp-
tion coefficient in the NIR wavelength region. Addition-
ally, the tissue auto fluorescence is minimal in this wave-
length range, which results in a higher signal/background
noise ratio. Fluorescence imaging in the visible range can
penetrate only up to 1–2 mm in depth, whereas NIR light
MOLECULAR IMAGING PROBES
Figure 8. Molecular probes for near-infrared imaging.
can penetrate up to several centimeters. Indocyanine
green is one Food and Drug Administration–approved
agent that has been used for ophthalmic retinal angiogra-
phy. Consequently, there is an increasing trend to attach
biomolecules with fluorescent dyes in the NIR range.
The NIR fluorochromes are coupled to antibodies or
peptides or a nonpeptide small molecule–targeting agent
that have specificity for biological targets (Fig. 8). Tar-
geted NIR fluorescent agents have been developed for
measuring apoptosis, osteoblastic cellular activity, angio-
genesis, receptors, and tumor-specific agents (13–15).
They have been targeted toward measuring phosphatidyl-
serine for apoptosis, somatostatin receptor, folate recep-
tors, and antitumor monoclonal antibodies for cancer.
Quantum dots (QDs) have been studied for some time
for their potential use in optical imaging. QDs are semi-
conductor crystallites that can be made into colloidal par-
ticles. These QDs can be tuned to absorb light of one
wavelength and emit light at different wavelengths de-
pending on the composition and size of the QDs. The
limitations of these QDs are their colloidal instability,
unwanted distribution, in vivo instability, and toxicity.
Various QDs that are coated with polymers have been
synthesized for their potential use in imaging. Recently,
Dubertret et al (16) have synthesized cadmium selenide
coated with zinc sulfide QDs that are encapsulated in
phospholipids micelles for in vivo imaging.
Smart Probes
Smart probes have been developed for optical imaging
and MRI. These probes have the advantage of having
nearly no signal and are undetectable in their native in-
jected state in vivo, but once activated, provide a strong
signal at the site of action and have a high signal-to-noise
ratio. The various physical mechanisms to activate the
probes include temperature and pH of the microenviron-
ment. Biochemical mechanisms are based on activation
by enzymes. Such probes have been used mainly to local-
ize enzymatic activity and function. Smart probes have
been developed for optical imaging. Many NIR smart
probes have been developed recently for imaging protease
activity. These probes are engineered so that they are
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 DANTHI ET AL
Figure 9. Cleavable molecular probes.
dark in their native quenched state and fluoresce only on
interaction with their specific proteases. The protease
cleavage site is placed between the fluorophore and the
quencher and the fluorescence is quenched because of the
proximity to the quencher. In the presence of the appro-
priate protease enzyme, which recognizes the specific
peptide sequence, the substrate is cleaved enzymatically,
which separates the fluorophore from the quencher and
bright fluorescence can be detected in vivo. Activatable
probes have been used to image cathepsin B, K, and D;
caspase 1 and 3; matrix metalloproteinases 2, 9, and 13;
urokinase; and other proteases. Weissleder and coworkers
(17) have shown NIR optical imaging of proteases in can-
cer. Urokinase plasminogen activator (uPA) and uPA re-
ceptor have been shown to facilitate cancer cell invasion
and metastasis. Increased expression of uPA has been
found in various cancers. In this smart probe develop-
ment, the authors have used a peptide that is a substrate
for uPA. Multiple copies of this peptide are attached to
the ⑀-amine of lysine in a polylysine backbone and the
amine terminus of the clevable peptide is attached to the
NIR dye. To the peptide is also attached fluorescein next
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Reprinted from Academic Radiology, Vol 11, No 9, September 2004
to the carboxy terminus. Because of crowding, the fluo-
rescence of the NIR dye is quenched (Fig. 9). When the
enzyme cleaves the peptide, the NIR dye with peptide
fragment separates and the fluorescence signal intensity is
amplified.
Numerous variations have been made on the basic de-
sign of the protease activatable agents. These include at-
taching poly-L-lysine backbone with multiple methoxy
polyethylene glycol groups; the latter reduces the immu-
nogenicity of the carrier and increases plasma half life,
avoiding rapid clearance and increased tumor delivery.
Additionally, nanoparticles have been used as a substrate
for the attachment of fluorochromes.
MPs for Multimodality Imaging
Multimodality imaging involves imaging the same sub-
ject with two or more imaging modalities to obtain com-
plementary information. Recently, a great deal of effort
has been placed into building multimodality imaging in-
struments, resulting in an increased interest in the synthe-
sis of MPs for the new instruments. Multimodality imag-
ing can potentially provide both anatomic information and
 Reprinted from Academic Radiology, Vol 11, No 9, September 2004
Figure 10. Dual probe.
as well as functional, metabolic, or molecular information
simultaneously. Several groups have synthesized multi-
function probes for animal imaging. Recently, a dual
NIR/MRI probe (18) has been synthesized for imaging
animals with optical/MRI techniques. The authors have
attached a polylysine polymer to NIR dye (Cy5.5) on one
side and cross-linked iron oxide on the other side (Fig.
10). By injecting this dual probe in animals and imaging
with MRI and optical methods, the authors have com-
pared the visual presentation of brain tumors during sur-
gery with the multislice topographical capability of preop-
erative MRI.
Molecular Probe Development: An Example
For the development of efficient targeted MPs, the first
step is the identification of an in vivo molecular target
that is specific for the physiologic and or pathologic con-
ditions of interest and has to be expressed in large num-
ber. Molecular biology techniques such as genomics and
proteomics play a vital role in this first step. After the
target is identified and validated, the next step is identifi-
cation of a targeting agent. The targeting agent can be an
antibody, a protein, a peptide, or a synthetic small mole-
cule. Identification and preparation of specific antibody or
protein is achieved by rigorous biochemistry. Peptides
and peptidomimetic small molecules are designed and
synthesized by medicinal chemists using rational drug
design or by combined library screening of thousands of
compounds. After a lead compound is identified, it is op-
timized for its physical, chemical, and biologic properties.
After having identified the target and the targeting agent,
Figure 11. Flow diagram illustrating the steps in developing molecular imaging probes.
MOLECULAR IMAGING PROBES
the design of MP is based on the type of imaging tech-
nique that will be used (Fig. 11). The targeting agent can
be labeled with a radioactive atom for PET or SPECT
imaging, a paramagnetic ion for MRI, or with a fluores-
cent tag for optical imaging. Alternatively, large macro-
molecules (such as nanoparticles or dendrimers) can be
synthesized with a label that can act as a contrast agent.
As an example, integrin ␣v␤3 is used to explain the
process of MP development. Integrin ␣v␤3 are known to
be expressed in physiologic and pathologic conditions
(eg, cancer, rheumatoid arthritis, macular degeneration).
Various groups have synthesized probes that target inte-
grins for imaging angiogenesis. There are hundreds of
patents and thousands of publications that list several pep-
tides, peptidomimitics, or antibodies that selectively bind
to integrin ␣v␤3. Few of them are in human clinical trials
for inhibiting angiogenesis and in treating the underlying
pathologic condition. Several groups have synthesized
integrin ␣v␤3–targeted MPs for PET, SPECT, MRI, US,
and optical methods and will be discussed in the follow-
ing section.
Integrin-Targeted MPs for PET and SPECT.—As de-
scribed previously, PET MPs are radioactive compounds.
One of several positron emitters (18F, 64Cu, 13N, 15O, 60Cu,
66Ga, 76Br, 86Y, 94mTc, 124I) can be used to label the targeting
agent. Each of these radionuclides require a separate method
of preparation using a cyclotron and different synthetic
methods of attaching to the targeting agent. A cyclic peptide
(cyclo RGDfMeV) has been used in most of the PET MPs
targeted to integrins. This peptide, in most cases, has been
successfully labeled with 18F for PET imaging. The labeling
was accomplished using [18F]AcOF (19). This method of 18F
labeling can be done on peptides that contain phenylalanine
residue. The integrin-targeted cyclic peptide does contain a
phenylalanine residue; thus direct 18F labeling was under-
taken. In another study, similar cyclic peptide was attached
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 DANTHI ET AL
Figure 12. Integrin-targeted probes.
with a glycopeptide labeled with 18F is used to image by
PET (20).
Integrin-Targeted MPs for MRI.—One of the first
␣v␤3targeted tumor angiogenesis imaging agent was de-
veloped by Li and coworkers. They attached the
␣v␤3antibody LM609 to their nanoparticle system (Fig.
12), which also contained Gd chelated to diethylenetri-
aminepentaacetate. This targeted nanoparticles showed
angiogenic hotspots in the tumor that cannot be seen by
conventional MRI. When the nanoparticle did not contain
the targeting antibody, no contrast was seen in the tumor.
Another group (21) also has shown significant increase in
contrast enhancement using Gd-containing nanoparticles
with a small molecule integrin targeting agent.
Integrin-Targeted MPs for US Imaging.—Biotinylated
microbubbles were prepared by sonication of an aqueous
dispersion of perfluorocarbon gas with lipids and then
combined with avidin and then with biotinylated echista-
tin. The authors have shown that this type of targeted
microbubbles can be used to image by US and spatially
assess angiogenic responses that occur early in the devel-
opment of malignant gliomas.
CONCLUSION
In conclusion, if the target of interest for imaging is
identified and a targeting ligand is available, development
of MPs for specific molecular imaging is possible. Devel-
opment of MPs for molecular imaging will likely parallel
the development of therapeutic agents in the future.
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Reprinted from Academic Radiology, Vol 11, No 9, September 2004
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A Primer on Molecular Biology for Imagers:
VIII. Equipment for Imaging Molecular Processes1
David M. Thomasson, PhD, Ahmed Gharib, MD, King C.P. Li, MD, FRCP(C), MBA
Molecular imaging (MI) in the broader sense is the char-
acterization and measurement of biologic processes at the
cellular and molecular level to elucidate various disease
processes (1,2); however, for radiologists, MI can be con-
sidered as the evolution of clinical diagnostic imaging
techniques toward a more specific characterization of dis-
ease processes and gradually toward a personalized level
(3). As genetic engineering progresses, MI may also be a
way of following this process in vivo for improved pre-
symptomatic disease detection and for providing tools to
follow progression of the disease or response to various
therapies (4). Since the inception of MI, its utility and
development in clinical diagnosis and therapy monitoring
has continued to evolve at a rapid pace (5–7). Just as vi-
sual observations of morphologic tissue changes on con-
ventional clinical imaging techniques gave way to the
physiological examinations of inner organs through the
practice of radiology, novel MI applications will further
develop the clinician’s ability to detect disease at earlier
stages to more accurately follow the patient through the
disease process.
Further advances in clinical MI seek to extend disease
characterization by harnessing rapid advancements of ex-
isting imaging technologies with the simultaneous evolu-
tions in the field of molecular biology for the study of
disease processes (8 –12). With current technology, direct
in vivo observation of molecules is beyond the scope of
most noninvasive imaging technologies. However, many
diagnostic studies can extend by inference down to the
molecular level those events that may be used for a better
Reprinted with permission from Acad Radiol 2004; 11:1159 –1168
1 From the National Institutes of Health, Clinical Center, Building 10, 10
Center Drive, MSC 1182, Bethesda, MD 20892-1182 (D.M.T., A.G.,
K.C.P.L.). Received July 16, 2004; accepted July 19, 2004. Address corre-
spondence to K.C.P.L. e-mail: kingli@nih.gov
© AUR, 2004
doi:10.1016/j.acra.2004.10.009
Chapter VIII
understanding of the molecular basis of the disease pro-
cess, improved sensitivity and specificity in the detection
of disease, and the clinical efficacy of various treatment
regimes. The goal of this report is to review some of the
MI applications in order to build a foundation for under-
standing the role of the imaging modality, discuss com-
mon imaging constructs and some of the basic physics
involved in the imaging strategies, and to summarize the
imaging characteristics of the imaging modalities with
respect to their niche role in the evolving field of molecu-
lar imaging.
The clinical practice of radiology started with noninva-
sive morphologic studies of tissues by exposing plain film
to x-rays and interpreting the subsequent shadowgram.
This noninvasive observation evolved through various
paths based on technical and physical innovations in x-ray
production and detection such as computed tomography
(CT), in which simple x-ray absorption is coupled with
projection reconstruction; nuclear decay detected by sin-
gle photon emission computed tomography (SPECT) and
positron emission tomography (PET) nuclear studies; dif-
ferential sonic absorptions/reflections in modern ultra-
sound (US); and nuclear spin relaxation in magnetic reso-
nance imaging (MRI). Table 1 is a simple summary of
the basic characteristics of each of these diagnostic tools
(9). Although all of these imaging modalities have the
ability to demonstrate morphologic characterization, the
practice of radiology has always combined this observed
morphology with pathologic characterization of diseases
to provide the most useful diagnostic information. We are
now seeing more publications tying MI to clinical dis-
ease.
Nuclear imaging studies were the first diagnostic ex-
ams that moved from mere morphology to a more tissue-
specific disease characterization. Here, the relatively poor
spatial characterization of the radiotracer is relatively un-
important compared with the physiologic process that lo-
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Table 1 tion of spatially localized metabolites—and physiologic

General Specifications for the Various Imaging Modalities
imaging such as perfusion and diffusion. However, this
Available for Clinical Molecular Imaging
review will focus on equipment used for those strategies
Modality Spatial Resolution Sensitivity that target cellular molecular events.
Positron emission
tomography 1–3 mm 10⫺11–10⫺12 mole/L
Single photon UNDERSTANDING THE TASKS OF MI
emission computed APPLICATIONS
tomography 5–10 mm 10⫺10–10⫺11 mole/L
Magnetic resonance MI has been defined as “spatially localized and/or tem-
imaging 10–100 ␮m 10⫺3–10⫺5 mole/L porally resolved sensing of molecular events in vivo”
Optical fluorescence 1–2 mm 10⫺9–10⫺12 mole/L (13). This definition sets up the necessity of understand-
Ultrasound 50–500 ␮m Not well characterized
ing the various technologies used to accomplish MI tasks.
Sensitivity and specificity ranges provide boundary conditions This definition also demonstrates the need to appreciate
for the various targeted agent applications. the fundamental limitations of each modality in terms of
where each technology fits into specific MI applications
(14,15). For clinical medicine, there are three main tasks
calized the radiotracer to that particular location in the that can be addressed with molecular imaging: disease
body. Figure 1 shows several molecular level targets that detection, pathology characterization, and the monitoring
have been used in nuclear medicine and, consequently, of various therapies.
are potential targets for the other imaging modalities. Re- A simple “detection” MRI protocol for localizing the
cent development of contrast agents that may be used in presence of potential tumors might include a MRI scan
conjunction with the other diagnostic modalities are now using, for example, a T2-weighted pulse sequence for
able to provide more relevant molecular level disease localizing suspicious regions within an organ. A molecu-
characterizations. Two additional areas of diagnostic im- lar imaging counterpart would be to inject a tumor-spe-
aging development include metabalomics—the quantifica- cific marker and then localize these with the appropriate

mRNA

DNA

Intracellular enzyme

Extracellular enzyme
Cell surface Cell surface

Figure 1. Potential targets for molecular imaging. Targeted contrast agents can be
chosen to study a variety of molecular processes by labeling imageable makers to spe-
cific cell sites.

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MR technique that is designed to “see” the specific
marker. An imageable molecular event is usually the
overexpression of a gene producing mRNA that results in
a specific protein production. This protein may end up in
the structure of the cell, its cell surface receptors, or an
enzyme. Any step in this process can provide a potential
target for molecular imaging (Fig. 1). In summary molec-
ular imaging uses these same basic imaging physics, but
includes molecular markers to extend the application to
molecular level events.
Molecular imaging in terms of targeted detection has
been implemented in diagnostic radiology departments for
some time through the use of tagged radiopharmaceuticals
in nuclear medicine. These studies have provided very
high specificity and sensitivity for the detection of various
diseases either throughout the body or within a given or-
gan system. The ability to tag different nuclear species on
a variety of metabolic markers has been very successful.
Molecular imaging in this context requires the use of a
radiolabeled particle that is specific to a given molecular
process, such as imaging endogenous gene expression
(16).
Unperturbed molecular environments may also be
assessed without targeted markers using suitable mo-
lecular level techniques, which may include local blood
flow, bulk perfusion (or the perfusion of specific mark-
ers), and bulk water diffusion at the cellular level. An-
other form of characterization would include non-inva-
sive chemical analysis, such as is possible with MR
spectroscopy.
COMMON IMAGING CONSTRUCTS
It is important to have a fundamental understanding of
imaging building blocks to evaluate equipment for molec-
ular imaging tasks. The fundamental imaging characteris-
tics of any acquisition modality can be broken into three
major constructs: spatial resolution, signal-to-noise ratio
(SNR) (contrast-to-noise ratio [CNR]), and temporal reso-
lution. These three constructs determine the sensitivity
and specificity of a specific imaging modality for achiev-
ing a given imaging task. In addition, two other factors
need to be considered: the safety of the imaging modality
and the availability and sensitivity of appropriate molecu-
lar probes. When one is considering which modality is
most appropriate for answering a given question, the rela-
tive strengths and weaknesses of these constructs for each
modality guide the appropriate decision.
Spatial resolution is the ability to resolve adjacent
points in an imaging plane and is quantified by the modu-
lation transfer function, or the more common parameter
“line pairs per millimeter.” Recently, resolution measures
for three-dimensional (3D) data sets are described in
terms of microliter volume sizes. For digital imaging sys-
tems, it is important to distinguish between pixel resolu-
tions and actual image acquisition resolution because it is
possible to have a very high pixel matrix without the un-
derlying acquisition resolution to support the perceived
matrix resolution. CNR is the term used to describe the
ability to distinguish a particular signal against a back-
ground of similar signals. Without sufficient contrast be-
tween two points, it would be impossible to distinguish
spatial boundaries irrespective of what the underlying im-
age resolution can support. The modulation transfer func-
tion is used to describe this behavior. Another critical
characteristic for studying dynamic behavior is the tempo-
ral resolution of a given image acquisition. In some mo-
lecular imaging tasks, this dynamic behavior is the key to
answering a given question. Imaging systems have a spe-
cific CNR behavior that is very strongly coupled to the
image acquisition time; together, these determine the sen-
sitivity and specificity of the given modalities in studying
dynamic processes requiring high temporal resolution.
CNR per unit time is a useful parameter for comparing
various systems.
The terms sensitivity and specificity of imaging sys-
tems are parameters based on the previous three image
“building block” characteristics used to describe the mo-
dalities’ ability to answer a particular MI question. If one
understands that imaging is primarily “signal” mapping,
then it is easy to see how the term signal can take on a
range of different characteristics. In conventional radio-
graphs and CT images, the signal is primarily dependent
on the absorption of transmitted x-rays; therefore, under-
standing resolution and signal to noise is very straightfor-
ward with respect to answering morphologic questions.
However, in a nuclear medicine exam in which the “sig-
nal” is nuclear emission, this answers a very different
signal mapping question: where and how the atom arrived
at a given location and how to best acquire the signal
from this radionuclide. However, as suitable contrast
agents are employed, this where-and-how question may
also be interrogated with CT imaging.
Based on these fundamental imaging characteristics,
we can now look more closely into the particular
strengths and weaknesses of each imaging modality for
determining the most appropriate tool for specific molecu-
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lar imaging applications. Table 1 shows a brief overview
of the various modalities used in current MI applications.
X-Ray and CT
Conventional radiographic and CT imaging systems
are based on the differential absorption of low energy
x-rays as they are attenuated during their transmission
through the body from source to detector. Advances in
diagnostic x-ray imaging followed the progress of techno-
logic innovations in both detector systems and computer
processing. Roentgen’s first two-dimensional projection
x-ray shadowgram has evolved from a simple differential
x-ray absorption/detection process to incorporate ad-
vanced film-screen systems with higher detection quantum
efficiency, light amplification systems as found in modern
fluoroscopy systems, and digital systems where x-ray ab-
sorption is stored on a solid state detectors. Autoradiogra-
phy, another film system, continues to be useful for spa-
tial localization in ex vivo samples (17); however, in
vivo– based MI requires the more sophisticated 3D imag-
ing strategies.
Modern CT, in which either gas-based or solid-state
detectors are coupled to sophisticated reconstruction algo-
rithms translating multiple lines of intersecting projection
data into 3D x-ray absorption data sets has a role in some
of the more morphologically based MI applications. In
addition, CT development with multirow detector systems
significantly improve the temporal resolution with little
compromise in spatial resolution. With these advances in
speed, a basic morphologic tool has been transformed into
one capable of physiologic measurements. Nonionic io-
dinated contrast agents have been developed for use with
multirow detectors systems to facilitate this process as a
clinical tool for evaluating organ perfusion.
The imaging characteristics of multirow detector CT
make it most suitable for high spatial resolution studies
and for applications requiring a corresponding high tem-
poral resolution as well. The drawbacks to CT applica-
tions are the limited soft-tissue contrast sensitivity and the
concomitant deleterious x-ray energy absorption. Because
the attenuation coefficient of the x-rays is due to interac-
tions in the electron cloud of irradiated atoms, there is
little contrast sensitivity because tissue is mostly water.
CT scanners are calibrated in Hounsfield units and there-
fore may be used in a quantitative manner for most tissue
characteristics. However, the primary limitation to CT
imaging is that X-rays must be absorbed or scattered to
reflect attenuation and this results in a dose (energy im-
parted) that must be delivered to the tissue. It is well
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known that high x-ray doses have a deleterious effect on
biologic tissues and to obtain good image quality, more
signal equates directly to more dose.
The trend in technologic advancements is toward a
higher number of detector arrays as well as a higher de-
tector quantum efficiencies. More detector arrays allow
simultaneous acquisition of several slices of data per rev-
olution of the x-ray detector pair. This significantly im-
proves temporal resolution. Solid-state detector arrays
with faster temporal response are also used to increase the
CNR for the given system. With these advancements, it
will be possible to further improve the temporal resolu-
tion while maintaining safe radiation dose levels for the
patients.
The primary current application for CT in basic sci-
ence MI is based on the high spatial resolution; this has
been implemented in specially designed micro-CT for
phenotyping transgenic mice. However, with the increased
speed and reduced dose of newer systems, physiologic
perfusion imaging has emerged as a possible MI applica-
tion (18). CT scanning of various molecular weight con-
trast agents with suitable pharmacokinetic models is also
being done to study permeability of some liver lesions
(19,20). Another increasing application of CT is its role
when coupled to PET imaging. This initial combination
was designed to overlay PET images on to high-resolu-
tion morphologic CT images.
Nuclear Isotope Imaging
Nuclear isotope imaging is the most prevalent form of
molecular imaging because of its ability to spatially local-
ize biologically relevant processes. Research applications
in isotope imaging have been steadily evolving for the
past 25 years (21). The basic physics of nuclear medicine
shares some of the same basic detection principles as x-
ray absorption processes. The fundamental difference be-
tween diagnostic x-ray transmission imaging systems and
nuclear medicine is the location of the radiation source
and the ability of the detector to discriminate the energy
of the emitted radiation. X-rays are based on differential
transmission of external ionizing radiation, whereas nu-
clear medicine is based on differential radioisotope uptake
within a particular organ system using radionuclides of
different emitted gamma energy. This focus on targeting
disease processes is the major advantage of radionuclide
studies for clinical applications. With localized sources,
the detector is only responsible for mapping the source
location and, with standard nuclear imaging as well as
 Reprinted from Academic Radiology, Vol 11, No 10, October 2004 EQUIPMENT FOR IMAGING MOLECULAR PROCESSES
SPECT, the ability to determine the energy of the emitted
radiation.
Detection of emitted gamma rays can be achieved by
various means. In some of the first nuclear systems, a
simple Geiger counter positioned at the neck could mea-
sure radioactive iodine administered intravenously. More
sophisticated multidetector systems using scintillation
cameras equipped with various collimator systems
evolved to better spatially localize various radioisotopes.
In all detector systems, the absorbed photons are con-
verted into energetic electrons whose signal is amplified
to produce a given measurable electrical signal. Weak
electrical signals are filtered and amplified to further im-
prove image contrast to noise. An amplification example
is the well known photomultiplier tube, which is based on
the absorption of high-energy photons in a phosphor. This
absorption results in the emission of an electron that sub-
sequently cascades into an avalanche of electron current.
One of the major advantages of a nuclear imaging system
is its semiquantitative nature. There is a direct correlation
between detected events and the concentration of the ra-
diotracer in the specific organ. Quantitation, however,
requires calibration factors and accurate attenuation cor-
rection factors that may be determined by various means
(22).
At the same time plain film x-rays were evolving to
modern CT technology, the ability to measure radiation
interactions based on radionuclide decay followed similar
progress. First nuclear medicine detectors were designed
to produce two-dimensional projection images, but later
were coupled to rotating multidetector systems SPECT to
extend this to 3D imaging. With 3D imaging, a better
co-localization of detected events and the organ of inter-
est are achieved.
PET is similar to standard SPECT regarding its ability
to three-dimensionally register radionuclide concentrations
with the exception that PET radionuclides are positron
emitters that necessitate coincidence detection. Coinci-
dence detection improves contrast to noise through better
background suppression. One disadvantage to PET imag-
ing is that typically, positron-emitting radioisotopes have
shorter half lives than other nuclear medicine radiophar-
maceuticals and, therefore, more difficult to handle with
respect to ease of production and distribution and radia-
tion safety. Another disadvantage is that all PET radioiso-
topes follow the same decay process; therefore, it is only
possible to follow one molecular species in a given imag-
ing experiment.
For both radionuclide imaging systems, the fundamen-
tal CNR is limited because of excessive absorption (radia-
tion exposure). This allowable exposure also limits the
detectable resolution. Resolution in standard nuclear im-
aging is based on collimator designs and is a tradeoff
with signal to noise (higher SNR with larger collimator
aperture but decreased resolution). The limit to spatial
resolution is based on the collimator technology coupled
to the previous CNR limits. PET is very different from
conventional nuclear studies due to coincidence detection
technology. Coincidence detection requires very fast tim-
ing characteristics in symmetric detectors. However, this
process has better background suppression of detected
events and can take advantage of different methods for
resolution improvement.
Several advances in nuclear detection are designed to
increase the resolution, sensitivity, and specificity. In
standard nuclear medicine studies, grid collimator designs
are the limiting factor for most clinical systems; however,
newer pinhole collimators can be used to improve resolu-
tion at the cost of SNR. More recent advances combine
these two designs to maintain the spatial resolution with
reduced loss in SNR. The 3D aspect of SPECT benefits
from improvements in collimator design, and sophisti-
cated tomographic reconstruction algorithms also continue
to be improved (23). Positron imaging has some unique
challenges based on coincidence detection and other lo-
gistical barriers, but because of its unique role based on
specific radiotracers, significant efforts are expended to
advance this technology (24).
Combining the attributes of two imaging modalities is
another MI strategy for improving detection. Advances in
software to merge imaging data sets will be important in
the success of this technique (25). The most well known
is the PET/CT combination in which the high spatial res-
olution of the CT scan is overlaid with the high specific-
ity of radiolabeled PET radiotracers. Figure 2 shows how
these technologies have been used to overlay a PET im-
age onto a corresponding head-and-neck tumor. Combina-
tion of these technologies can improve PET quantification
of radiotracers through attenuation correction as well as
partial volume correction.
Nuclear medicine radiopharmaceutical studies were the
first and continue to be the most prevalent method of cur-
rent radiology-based MI applications. Nuclear-based mo-
lecular-genetic imaging is one of the more prevalent ap-
plications (16,26 –28) and can be accomplished both di-
rectly and indirectly. Nuclear medicine applications
continue to develop in part because of the ability of label-
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Figure 2. Axial computed tomography (CT) image through tumor region of the
neck (A). Axial positron emission tomography (PET) image at the location corre-
sponding to CT image (B). CT image with PET overlay (C). Coronal PET image
showing distribution of radioisotope throughout the body (D).

ing a wide variety of biologically relevant molecules with
radioisotopes. Standard nuclear studies use 99mTc, 68GA,
and 82RB, which are easily produced in isotope generators
through decay processes. PET radiotracers such as 18F,
15O, and 11C are produced through cyclotron interactions
but are becoming more available through regional distri-
bution systems. Several clinically focused articles high-
light PET radiopharmaceutical studies that bridge the im-
aging modality from clinical to more cellular and genetic
events (29 –33).
ULTRASOUND
Ultrasound also has enjoyed significant technologic
innovation that enables the extension of simple mor-
phologic imaging to molecular imaging. Ultrasound
imaging is based on the detection of differential reflec-
tion of sound waves as they travel through tissue. Re-
flection coefficients are used to characterize the various
tissues by their ability to reflect, absorb, or scatter the
incident US pressure wave. Initial systems used single-
transmit/detect piezo-electric crystals rastered across
the body to “paint” a picture of calibrated reflection
coefficients. The contrast is determined by the differ-
ence in the reflection coefficients and is best appreci-
ated in terms of transition zones between air and fluid.
Fluid transmits US easily, air reflects US almost com-
pletely, and soft tissues have reflection characteristics
between these two extremes. It is also possible to char-
acterize the shadows produced in this transmission/
reflection process.
Molecular imaging US applications are primarily based
on the use of contrast agents designed in the form of
“bubbles.” Various molecular structures may be labeled to
the outside of this bubble to make it target specific (34).
US imaging with contrast “bubbles” are based on two
interactions. If the wavelength of the transducer is
matched to the properties of a given contrast agent bub-
ble, a specific signature can be measured (35,36). Alterna-
tively, the US probe may receive signals based on the
collapse of the bubble during US interrogation.

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Figure 3. Ultrasound image of thrombus in the rabbit after administration of biotinyl-
ated-CD41 or saline followed by BG0470 bubbles. (Figure adapted with permission;
from Fig. 1 of reference 38.)
The boundary conditions for resolution are determined
by the frequency of the US transducer; in general, higher
frequency transducers will provide better resolution. An-
other boundary condition for US is the penetration depths.
Because US waves are both reflected and absorbed in
tissue, there is a gradual loss of SNR as a function of
depth. At higher frequencies, however, there is a concom-
itant increase in absorbed power that translates to de-
creased penetration and tissue heating that must be con-
sidered in patient safety.
Next-generation US systems are based on arrays of
smaller transducers simultaneously coregistered to pro-
duce higher spatial and temporal resolution images. This
coupled with advances in digital signal processing tech-
nology resulted in real-time 3D imaging. With suitable
postprocessing of temporally sampled reflected waves, US
was able to measure flow using a Doppler effect. This has
been extended from simple single line vessel flow studies
to more sophisticated power Doppler studies that have the
ability to assess diffusion type tissue properties using
color mapped spectral analysis of reflected waves (37).
Primary molecular imaging applications for US include
tissue characterization with specifically designed contrast
agents as well as ultrasound biomicroscopy, which is be-
ing used to study tumor angiogenesis (12). Figure 3
shows how targeted bubbles are used to label thrombus
(38). In addition, one further interesting role of ultrasound
takes advantage of its ability to cause a mechanical inter-
action (heating or mechanical stress) at specific localized
target molecules to either study MI events or initiate MI
processes such as bubble destruction with subsequent re-
lease of targeted therapeutic agents (39).
Optical Imaging
Light microscopy is the primary tool for biologic im-
aging at the cellular level and most other imaging tools
use the results from histopathology labs as the gold stan-
dard to compare their results. This form of imaging has
very high temporal and spatial resolution in addition to
the good SNR and has been extended by various process-
ing strategies to accommodate the changing needs of the
biologic researcher (40). However, light optics have limi-
tations when they are extended down to the molecular
level. Here, too, optical imaging must resort to more indi-
rect detection strategies to characterize molecular events.
The extension of this optical imaging tool to molecular
imaging may be accomplished in several fashions, atomic
force microscopy (41,42), direct infrared imaging, biolu-
minescence imaging, fluorescence-epifluorescence imag-
ing (43,44), and confocal optical imaging (45). The use of
laser light sources coupled to these techniques extends the
range of possible optical MI applications (46). Optical
scanning holography, which was previously limited to
thin sections, is being developed to accommodate more
biologically relevant samples (47).
The common process in many optical techniques is
camera technology. A camera is necessary to convert
photons to storable signal through suitable optics. Cam-
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Figure 4. Ductal carcinoma (a), function sagittal magnetic resonance (MR) image after
GD contrast enhancement passing through the center of the cancerous lesion. (b)
Coronal diffuse optical tomography image perpendicular to the plane of the MR image
in (a). (c) Functional MR coronal reslicing of the volume of interest with the same di-
mensions as in (b). (Figure adapted with permission; from Fig. 3 of reference 50.)

eras use various technologies to both translate photon en-
ergy into electrical signals that are subsequently mapped
and stored into digital arrays with filtering technologies to
maximize CNR. Cameras can be made sensitive to differ-
ent wavelengths by using a variety of atomic electron
absorptive media and may include both amplification
strategies as well as noise suppression techniques to boost
signal above background noise. Charge-coupled device
(CCD) cameras use an absorption process to convert light
photons at specified wavelengths to a measured signal.
CCD cameras use both optical filters and various input
phosphors to preselect specific wavelengths. Two modes
of optical imaging include reflection or transmission, and
in both the characteristics of the light absorption and scat-
ter may be used to characterize the medium investigated.
Major advantages of optical techniques are the high
relative SNR and the high temporal resolution of optical
events. Resolution is typically limited because of digital
resolution in the camera, but camera technology is im-
proving dramatically and typical CCD cameras are in the
megapixel range. To a lesser extent, high-contrast spatial
resolution is also controlled by standard lens optics. In
MI applications the optical probes concentration dictates
CNR. In vivo optical imaging strategies are primarily lim-
ited based on the absorption of light as it travels through
the body. Although-high frequency (short wavelength)
light suffers less absorption, it is still limited to either
superficial tissues or requires surgical exposure or inser-
tion of camera lens or optical fibers into tissues for visu-
alization of deep-seated lesions.
Based on the rapid popularity of consumer digital cam-
eras, CCD detector arrays have been rapidly transformed
to higher and higher matrices with consequent higher dig-
ital resolution. Here too the hardware and software tech-
nology to process these data had to keep up with the in-
creasing speed of data acquisition of larger arrays. With
these light-measuring arrays, it is also possible to inter-
pose a scintillator to produce both optical as well as nu-
clear detector for some MI applications. Advances in both
light source as well as detectors are necessary to follow
various optical probes and new designs are necessary to
overcome the depth limitation of this technology (48). In
addition catheter-based probes for the study of internal
organs are being designed to be small and flexible so that
they can be used for more localized procedures. Addi-
tional processing strategies are being developed to charac-
terize some of the temporal characteristics of optical con-
trast agents to even further elucidate MI processes (49).
Some proof of concept studies have been performed to
show that greater optical penetration is achievable with
near-infrared portion of the optical spectrum. Figure 4
shows how the indocyanine green can penetrate breast
tissue (50). To improve the 3D localization of labeled
near-infrared dyes, dual-wavelength subtraction “thermo
acoustic computed tomography” techniques have been
developed (51) as well as fluorescence-mediated tomogra-
phy techniques (52,53).
Along with innovations in the detectors associated with
optical imaging are the developments in optical probes.
Quantum dots are a new technology for MI applications
in ex vivo applications but may be developed for in vivo
applications (36). These are small tunable crystals that
have signal-to-noise and resolution properties applicable
to cellular level investigations (54).

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Optical imaging is a versatile tool that extends to
many applications. One of the first digital optical methods
in medicine is direct infrared imaging, which can be used
to localize active physiologic/metabolic processes based
on heat generation. It has been used for cancer detection
and blood flow processes. It is primarily limited to super-
ficial regions due to the absorption of infrared wave-
lengths in tissue. The temporal resolution is good, but the
spatial localization is limited to both the absorption as
well as scatter. Advances in temporal resolution are possi-
ble through large area Fourier transform infrared spectro-
scopic imaging (55).
Epifluorescence is another strategy for optical imaging
based on the stimulated emission of fluorophores. One
example, fluorescein, when bathed in short-wavelength
blue light, emits a longer wavelength green light that can
be separated using a chromatic reflector. This has the ad-
vantage of high signal-to-background data but suffers
from the previously mentioned light detection processes
in tissue.
Confocal optical imaging is a strategy to obtain 3D
images of fluorophores by “out of focus” rejection of
emissions at given depths. In this manner, a sample may
be optically scanned to produce a 3D representation of a
given fluorophore distribution. Another technique used in
MI is optical coherence tomography. This technique is
analogous to clinical US imaging; whereas differential
reflection of ultrasonic waves are used in US, in optical
coherence tomography differential optical waves are used
in a similar manner to produce images. Optical coherence
tomography techniques can be used in catheters to charac-
terize endothelial plaques as well as in some gynecologic
applications (56).
MRI
Clinical MRI is based on the interrogation of tissue
water protons through the differential population of pro-
ton spins when a sample is placed in a large magnetic
field. Protons in the body when placed in a large mag-
netic field are separated into two spin-state populations
and when this differential population of spins is subjected
to a radiofrequency (RF) pulse at the proton resonance
frequency, the differential population of spins is perturbed
away from this new equilibrium state. However, because
the spins are still in the main magnetic field, they subse-
quently “relax” back to the original differential population
with a time-constant proportional to their local magnetic
environment. The signal produced in an MRI experiment
is due to the transitions in these two spin-state popula-
tions and is based on the “relaxation” of this differential
population. Spin mapping is based on applied magnetic
gradient fields that alter the resonant frequencies for the
spin transitions. Magnetic resonance spectroscopy is an-
other MR-based technique available for the study of mo-
lecular imaging. The protons on a molecule have a unique
signature that can be discerned in a magnetic resonance
spectroscopy experiment. Because of the low abundance
of these protons in biologic systems, the SNR is quite
low, but in the case of some nuclei, the SNR can be suf-
ficiently above background to enable MI studies (57).
The most important physics to appreciate with MRI is
its very low sensitivity with direct detection methods.
MRI is more sensitive and specific when used in an indi-
rect detection mode by watching the influence on tissue
relaxations resulting from either sequence parameters de-
signed to capture signal at specific points in the relaxation
curve or with the use of contrast agents such as the para-
magnetic atoms gadolinium or iron oxide. The fundamen-
tal technical limits to resolution and SNR are based on
gradient strengths and radiofrequency RF coils in con-
junction with main magnetic field strengths. In both of
these areas, technology has quickly approached the limits
set by physiologic events. The primary limitation to gra-
dient strength is the rate of change of the gradient field
db/dt. At high db/dt values, one can experience peripheral
nerve stimulation. This physiologic effect currently limits
standard clinical scanners with existing gradient perfor-
mance. Although the SNR can be increased with higher
main magnetic fields, it can also be increased with more
localized RF coils or coil array systems; here too, physio-
logic constraints come into play as smaller local coils are
limited by the depth of view.
As the importance of MI becomes more apparent MRI
technology is being developed to address these needs
(58). Clinical scanners have arrived at a compromise for
routine use at 3.0 Tesla and approximately 40 – 60 mT/m
(10 e-3 Tesla per meter) gradient strengths. There are
significant developments in array coil technology that,
when coupled with advanced postprocessing strategies,
can improve the temporal resolution without significant
decrease in SNR. Other advancements in MR software
are designed to better characterize the signal obtained
from the MRI data such as with diffusion tensor images
from multivector data sets or better postprocessing of in-
jected contrast dynamic data with more appropriate math-
ematical models of tracer transport. The use of endoge-
nous tracers in MRI has also been developed to character-
ize molecular processes. Recent developments in
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 THOMASSON ET AL Reprinted from Academic Radiology, Vol 11, No 10, October 2004

Figure 5. Magnetic resonance images of V2 carcinoma in the thigh muscle of a rabbit

and subcutaneously before (a) and at 24 hours (b) after LM609-labeled paramagnetic
nanoparticle injection. (b) Magnetic resonance images of isotype-matched controls. No-
tice the difference enhancement patterns seen in the thigh tumor model as compared
with the subcutaneous tumor model. (Figures adapted with permission; from Fig. 1 of
reference 61.)

hyperpolarized nitrogen and carbon nuclei have been used
in MRI molecular imaging (59).
MI in its broadest sense includes many of the func-
tional MRI applications found today on clinical scan-
ners. One aspect of molecular imaging is the interroga-
tion of the local environments, such as tumor angio-
genesis, diffusion, flow, temperature, and chemical
compositions. Current scanners have the ability to ac-
quire and process data to study these molecular envi-
ronmental events (60). Additionally, MRI applications
are being developed similar to nuclear medicine iso-
tope studies. Suitable tailored acquisition techniques
can be used for detection of targeted contrast agents.
Figure 5 shows how specific MR contrast agent– based
targeted molecules have been developed to detect an-
giogenesis in tumor models (61). Additional applica-
tions include the study of antibody/antigen processes
such as endothelial proinflammatory antigens (62,63),
imaging cell membrane components such as membrane
phospholipids (64), and the localization of cancer anti-
gens such as anticarcinoma monoclonal antibodies (65).
GLOSSARY
Attenuation coefficient: Exponential coefficient that
describes the interaction probability of x-rays with the
media they traverse.
Coincidence detection: Process by which positron
emitting radiations may be localized based on the simulta-
neous emission/detection of two 511 keV photons emitted
at 180 degrees apart in an internally administered radio-
isotope.
Collimator: High attenuation grid structure that only
transmits x radiation or gamma radiation from a specific
direction; used to localize in a planar fashion the source
of administered radioisotopes and consequently defines
the intrinsic spatial resolution of nuclear medicine imag-
ing equipment.
Contrast: Signal difference between two adjacent
points in an imaging plane; typically used with respect to
spatial resolution.
Detector quantum efficiency: Efficiency with which
absorbed x or gamma radiant energy is converted to elec-
trical signal in x-ray– based imaging systems.
Hounsfield scale: Image display scale value where
⫹1000 equals air and ⫺1000 equals dense bone; related
to attenuation coefficients for biologic materials.
Modulation transfer function: Parameter used to
characterize how well an imaging system can reflect sinu-
soidal varying input functions; typically presented as a
range of input frequencies.
Nuclear magnetic resonance: An exchange of radio-
frequency energy at a specific radio frequency determined
by the nuclear spin of the atomic nucleus and the mag-
netic field in which it resides.
Piezoelectric : Property of ultrasound transducer crys-
tals that converts electrical energy to mechanical energy
and vice versa.
Pixel: Digital imaging term meaning picture element
that describes the display resolution of a given imaging
system.
Radioisotope: Unstable isotope that spontaneously
emits radiation to form more stable isotopic form.
Reflection coefficient: Ultrasound parameter that de-
scribes the energy of the impinging ultrasound wave that
is reflected back to the ultrasound receiver.
Relaxation times: Nuclear magnetic resonance time
constants that quantify the longitudinal magnetization re-

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 Reprinted from Academic Radiology, Vol 11, No 10, October 2004 EQUIPMENT FOR IMAGING MOLECULAR PROCESSES
turn to equilibrium after perturbation by external magnetic
or radiofrequency fields T1; or the return to equilibrium of
the transverse magnetization T2.
Resolution: Parameter used to characterize an imaging
system in terms of its ability to convey information; spa-
tial resolution for the ability to resolve two adjacent struc-
tures, temporal for the ability to resolve two imaged
events in adjacent points in time.
Spin density: The number of resonating spins per unit
volume that determines the strength of the nuclear mag-
netic resonance signal.
Ultrasonic: Mechanical wave frequency spectrum gen-
erated by ultrasound imaging and therapy equipment.
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A Primer on Molecular Biology for Imagers:
IX. How to Become a “Molecular Imager”1
This is the final article in our series on molecular biology
specially written for imaging scientists. We believe that
this series of articles can be used by all imaging scientists
new to the field of “molecular imaging” as a stepping
stone toward this exciting scientific area. For many prac-
ticing radiologists and clinical imaging scientists, the first
question that may arise is why should they become mo-
lecular imagers?
To address this question, we have to examine what
makes imaging such an important part of medical practice
today. To be of clinical value, a diagnostic test needs to
provide useful information that would affect therapeutic
decisions. A diagnostic test decoupled from therapeutic
decisions will eventually become obsolete. Medical imag-
ing is providing mainly morphologic information that is
critical for patient care today. However, with the approval
of Trastuzumab (Herceptin, Genentech, Inc., San Fran-
cisco, CA) in 1998 for the treatment of HER2-positive
metastatic breast cancer and imatinib mesylate in 2001
(Gleevec, Novartis, Inc., Basel, Switzerland) for the treat-
ment of chromic myelogenous leukemia with a bcr-abl
translocation, it is obvious that the era of molecular thera-
peutics is upon us. To decide whether Herceptin is going
to be effective in a patient, it is no longer sufficient to
know the histologic classification and the stage of the
tumor. It is critical to know whether the breast cancer is
HER2-positive or not. As the development of molecular
therapeutics accelerates the growth of molecular diagnos-
tic testing is also increasing at a brisk rate estimated at
30%–50% per year (1). How the practice of “medical
Reprinted with permission from Acad Radiol 2004; 11:1
1 From the Department of Radiology and Imaging Sciences, Clinical Center,
National Institutes of Health, Bldg. 10 1C626, 10 Center Drive MSC 1182,
Bethesda, MD 20892–1182. Received August 9, 2004; accepted August 10,
2004. Address correspondence to K.C.P.L. e-mail: Kingli@nih.gov
© AUR, 2004
doi:10.1016/j.acra.2004.10.010
Chapter IX
King C.P. Li, MD, FRCP(C), MBA
imaging” is going to change in the era of “molecular
medicine” and “personalized medicine” is any one’s guess
at this point. However, it should be obvious that all
medical practitioners should have at least some basic
knowledge of molecular biology to keep pace with the
tremendous changes in molecular diagnostics and thera-
peutics. This is precisely the first step in becoming a
molecular imager, which I would define as “a practitio-
ner of medical imaging in the era of molecular medi-
cine.” Obviously, this definition is designed to be
vague and all inclusive but this is precisely what we
need to do so that we don’t get tunnel vision and the
field of molecular imaging can enjoy the rapid growth
similar to molecular diagnostics testing. How then can
one become a molecular imager? I believe it can be
divided into four different stages with increasing levels
of sophistication (Table 1). In the following sections I
will go into the details of each of the stages of devel-
opment and explain why it is important and how we
can achieve it.
STAGE 1: SEE MORPHOLOGY, THINK
MOLECULAR BIOLOGY
Radiologic-pathologic correlation has been the back-
bone of medical imaging in the past century. Much of our
interpretation of medical images today is based on the
wealth of knowledge accumulated through years of pain-
staking work by numerous radiologists and pathologists.
In fact, attending the 6-week course at the Armed Forces
Institute of Pathology and taking the American Board of
Radiology Examination are probably the only experience
common to the vast majority of radiologists trained in the
United States. A lot of time during our residency training
is spent on “pattern recognition” as well as memorizing
“gamuts” and “Aunt Minnie” findings. This has func-
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 LI
Table 1
Stages of Development of a Molecular Imager
Stage 1. See morphology, think molecular biology
Stage 2. Combine imaging information with molecular diagnostic
information
Stage 3. Obtain molecular information using imaging
Stage 4. Personalize treatment using combined molecular
imaging and therapy
tioned very well as evidenced by the rapid growth of
medical imaging examinations done worldwide and will
remain the dominant practice in the near to intermediate
future. However, with the explosion of knowledge in mo-
lecular biology our understanding of health and disease is
rapidly evolving in the genomic era. Imaging scientists
should begin thinking about the molecular basis of physi-
ologic and disease processes. This series of articles is
designed to help imaging scientists unfamiliar with mod-
ern molecular biology to learn the jargon and enough ba-
sic knowledge to enable them to keep current with bio-
medical advances by following the scientific literature.
Biomedical information is now widely accessible even to
the lay public. It is now common to hear “breakthrough
developments” in biomedicine through prime-time news.
The onus is on medical practitioners to know more about
these developments than our patients. Radiologists have
always been able to keep up with new knowledge. Molec-
ular biology is much closer to our core medical training
than magnetic resonance imaging physics. So, we should
be very optimistic that most practicing radiologists can
achieve this stage of development in becoming a molecu-
lar imager.
STAGE 2: COMBINE IMAGING
INFORMATION WITH MOLECULAR
DIAGNOSTIC INFORMATION
When I was a radiology resident, a faculty member
kept telling us during our film interpretation sessions to
remember that all the information was on the film and the
film wouldn’t lie. This, of course, is the wrong way to
interpret medical images. The more clinical, laboratory,
and other data that can be incorporated in the interpreta-
tion process, the more accurate the interpretation will be.
In fact, sometimes the pretest probability of a diagnosis is
so high that the imaging result can become almost irrele-
vant. In the genomic era, high-throughput tissue analysis
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Reprinted from Academic Radiology, Vol 11, No 11, November 2004
techniques such as functional genomics (2– 4), proteomics
(5–7), and tissue arrays (8 –10) are being developed to
provide a vast amount of data rapidly from any tissue
samples such as serum, urine, and biopsied tissue. Simi-
larly, multimodality imaging is becoming commonplace.
We have begun to develop ways to combine different
imaging datasets and hardware to exploit the synergy of
different imaging modalities. Combined positron emission
tomography–x-ray computed tomography, combined sin-
gle photon emission tomography– computed tomography,
combined angiography– computed tomography, and com-
bined angiography–magnetic resonance scanners are first
examples of this type of fusion imaging devices. The next
stage in the development of a molecular imager is to de-
velop the ability to objectively and systemically combine
this wide array of datasets to come up with the best inter-
pretation possible. To facilitate this, image-guided tissue
procurement followed by immunohistochemistry, laser
capture microdissection (11–13), functional genomics,
functional proteomics, and other tissue analysis should be
performed initially to gain insight into the complex bio-
logic processes. This will become the “radiologic-patho-
logic correlation” equivalent in the genomic era. Early
results in this area are very encouraging (14 –17). Obvi-
ously, new bioinformatics tools will need to be developed
before we can make sense of the large, complex data sets
generated by the various techniques (18).
STAGE 3: OBTAIN MOLECULAR
INFORMATION USING IMAGING
Although many of the concepts encompassed by mo-
lecular imaging have been practiced for decades in nu-
clear medicine and positron emission tomography, there is
still a paucity of clinically available molecular imaging
probes. This is not surprising considering that there are
more than 30,000 genes in the human genome but thera-
peutic agents used clinically today target fewer than 500
human gene products (19). However, the pace of discov-
ery of molecular targets, pathways, and compounds useful
for probing these targets and pathways may increase dra-
matically. This is due to three important technologic ad-
vances: namely, the sequencing of the entire human ge-
nome, developments in combinatorial chemistry, and ad-
vances in robotics plus informatics (20). The discovery of
chemical libraries with activities toward a large number
of molecular targets and pathways can accelerate the de-
velopment of molecular imaging probes. Starting with a
 Reprinted from Academic Radiology, Vol 11, No 11, November 2004 A PRIMER ON MOLECULAR BIOLOGY FOR IMAGERS
specific imaging probe, the temporal course and spatial
distribution of a molecular target or pathway modulated
during a physiologic or pathologic process can be deter-
mined. Using image-guided tissue analysis, the molecular
targets and pathways that change at the same time as the
molecular target imaged can then be determined. Using
this information new imaging probes can be developed.
Ultimately, a molecular imager can use an appropriate
cocktail of imaging agents to track noninvasively, in vivo,
in a temporally and spatially resolved manner several re-
lated molecular targets and pathways and see how they
change after different modulations. This is the next stage
of development for a molecular imager
STAGE 4: PERSONALIZE TREATMENT
USING COMBINED MOLECULAR IMAGING
AND THERAPY
In the past few years, there have been multiple publi-
cations that make the prediction that new techniques such
as pharmacogenetics, pharmacogenomics, and pharmaco-
proteomics will open the path to personalized medicine
(21,22). It has been stressed, however, that “personalized
medicine” should be conceptualized along multiple di-
mensions such as disease, environment, genes, medica-
tion, health care, and information (23). Molecular imaging
can certainly contribute to making “personalized medi-
cine” a reality. The most obvious way is to combine tar-
geted imaging and therapy using the same drug delivery
mechanism. For example, ibritumomab tiuxetan (Zevalin,
Biogen Idec, Inc, Cambridge, MA) is a radioimmuno-
therapy product for treatment of relapsed or refractory
low-grade, follicular, or transformed B-cell non–
Hodgkin’s lymphoma. An Indium-111–labeled Zevalin
scan is used for patient selection and dosimetry purposes
before administration of the therapeutic yttrium-90 –la-
beled Zevalin to the selected patients. Another example is
the use of a reporter gene to monitor gene expression in a
gene-therapy regimen. Similarly, nanoparticle systems
have been successfully used to carry contrast as well as
therapeutic agents to endothelial targets (24 –26). In the
future, it should be possible to use a “cocktail” of molec-
ular targeted imaging probes to determine the appropriate
combination therapy regimen for a specific disease in a
specific patient at a specific time. Imaging can be re-
peated to monitor the response and the therapeutic regi-
men can be modified based on the results. This will be
the final stage of development of a molecular imager in
the foreseeable future. Although this is still much inferior
to the Star Trek “medical tricorder,” it will be a giant
step toward the realization of “personalized medicine.”
SUMMARY
This is the conclusion of our series of articles entitled
“A Primer on Molecular Biology for Imagers,” and hope-
fully this will serve as the beginning of many readers’
journey in becoming a molecular imager. There is no real
barrier to achieving stage one of development, which re-
quires only acquisition of the knowledge base and the use
of this knowledge in our practice of imaging sciences and
also in our everyday life. We have made much progress
in building the foundation for further development of mo-
lecular imaging and we are optimistic that many of our
readers will participate in the revolution of biomedicine in
the genomic era.
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Subject Index
A Bragg’s law, S30
Acidic amino acids, S23
Activators, transcriptional, S15
Active site, of enzymes, in protein
synthesis, S30
Agarose gel electrophoresis, S37–
S38
Alkaline lysis method, for DNA
isolation, S36
Alzheimer’s disease, protein mis-
folding in, S30
Amino acids, S22–S24
Aminoacyl-tRNA synthetase, S25
Antennapedia factor, in transcrip-
tion, S15
Antibodies, S32–S33
Anticodons, S12
Anti-sense strand, in RNA replica-
tion, S13
B
B cells, antibody synthesis in, S32
Bacteria, transformation of, S39,
S41
Bacteriophage ␭ cro repressor, in
transcription, S15
BamH1 restriction endonuclease,
S37
Basic amino acids, S23
Basic domain, in transcription fac-
tors, S15–S16
Bioinformatics
in genomics, S55–S57
in proteomics, S67–S68
Bioluminescence studies, S41, S75–
S76
Bradford assay, for protein analy-
sis, S31
Branchpoint sequence, in post tran-
scriptional processing, S18
Breast cancer, genomics of, S57
C
C2H2 domain, in transcription fac-
tors, S15
Calcium phosphate, for transfec-
tion, S41
Cameras, for optical imaging, S85–
S86
Cancer
breast, genomics of, S57
molecular probes for, S76
ovarian, proteomics techniques in,
S69
Cancer Genome Anatomy Project,
S56 –S57
Cap (7-methylguanosine), in post
transcriptional processing,
S18
Carboxyl-terminal domain, in tran-
scription factors, S18
CDC6 protein, in DNA replication,
S8
cDNA, synthesis of, S46 –S47
CDT1 protein, in DNA replication,
S8
Centromeres, S6
CGAP (Cancer Genome Anatomy
Project), S56 –S57
Charge-coupled device cameras, for
optical imaging, S86
Choline, radiolabeled, for PET,
S72
[11C]Choline, for PET, S72
Chromatin, S5
Chromatography
high-performance liquid, in proteom-
ics, S62–S63
with mass spectrometry, S64
Chromosomes, DNA in, S5
Cloning, molecular, S37–S39
Coding strand, in RNA replication,
S13
Codons
start, S26
stop, S27
Collision induced decay, in mass
spectrometry, S63–S64
Column purification methods, for
DNA isolation, S36 –S37
Comparative genomics, S52–S53
Complementarity-determing re-
gions, of immunoglobulins,
S33
Computed tomography, in molecu-
lar imaging, S82
Confocal optical imaging, S87
Conformation, of proteins, S24
Conjugated proteins, S24
Constant region, of immunoglobu-
lins, S33
Contrast agents, for molecular im-
aging. See Molecular probes
Contrast-to-noise ratio, in molecu-
lar imaging, S81
Coomasie stain, for gel electro-
phoresis, S61
Cosmids, S39
Creutzfeldt-Jakob disease, protein
misfolding in, S30
Crick, Francis H.C., DNA discov-
ery by, S4
Crystallography, x-ray, in proteom-
ics, S65
S101
 SUBJECT INDEX VOLUME 11
Cyclic AMP receptor protein, in
transcription, S15
Cytosine, in DNA structure, S2,
S4 –S5
D Ethical, Legal, and Social Issues
Databases
in genomics, S55–S57
in proteomics, S67–S68
Degenerate genetic code, S6
Deletions, in mutation, S6
Deoxyribonucleic acid. See DNA
Dideoxyribonucleotide triphos-
phates, in DNA sequencing,
S42–S43
Differential gene expression analy-
sis, S20
Dimerization domains, in transcrip-
tion factors, S16
Direct infrared imaging, S87
DNA, S1–S9
detection of, S44
double helix form of, S2, S4 –S5
of eukaryotes, S5–S7
isolation of, S36 –S37
of prokaryotes, S5–S7
replication of, S7–S8
RNA action on, S13
sequencing of, S42–S44
structure of, S2, S4 –S5
vs. RNA structure, S10
in transcription, S14 –S15
triplet genetic code and, S6
DNA chip, in microarray technol-
ogy, S54 –S55
DNA helicase, S7
DNA ligase, in DNA replication, S8
DNA polymerase
in DNA sequencing, S42–S43
in replication, S8
DNA-binding domains, in tran-
scription factors, S15
Dopamine D2, detection of, for
PET/SPECT, S73
DOTA-octreotide, for PET, S72
Double helix form, of DNA, S2,
S4 –S5
Downstream promoter elements, in
transcription, S14
S102
Academic Radiology, Vol 11, Suppl 1, November 2004
Drugs, development of, proteomics
in, S69
E
Edman method, for peptide se-
quencing, S32
EGR-1 gene, Wilms’ tumor gene
and, S15
Electron microscopy, in proteom-
ics, S65
Electrophoresis, S37–S38
in proteomics, S61–S62, S66
two-dimensional polyacrylamide gel,
S62, S66
Electroporation, S41
Electrospray ionization, in mass
spectrometry, S62–S63
Elongation, in protein synthesis,
S27
ELSI (Ethical, Legal, and Social
Issues) program, in genom-
ics, S58
Encyclopedia of DNA Elements
(ENCODE) project, S22
Endonucleases, restriction, S37
Endoplasmic reticulum, protein
synthesis and, S28 –S29
Enhancer elements, in transcrip-
tion, S14
Epifluorescence, in optical imaging,
S87
Equipment, for molecular imaging,
S79 –S90. See also specific
modalities
applications of, S80 –S81
computed tomography, S82
magnetic resonance imaging, S87–
S88
optical imaging, S85–S87
radioisotope, S82–S84
sensitivity and specificity of, S81
spatial resolution in, S81
ultrasonography, S84 –S85
x-ray, S82
Escherichia coli
DNA replication in, S7
genome of, S5
polymerase of, transcription by, S11
RNA polymerase of, S13–S14
ESTs (expressed sequence tags), in
microarray analysis, S54 –
S55
program (ELSI), in genom-
ics, S58
Euchromatin, S6
Eukaryotes
DNA replication in, S7–S8
gene structure in, S7
genome of, S5–S6
ribosomal RNA in, S12
RNA polymerases of, S13–S14
transcription factors of, S15–S16
transcription in, S14 –S17
transfection of, S41
translation in, S24 –S27
Exons, S7, S18 –S19
Expressed sequence tags, in mi-
croarray analysis, S54 –S55
F
Fab fragments, of immunoglobu-
lins, S32
Fc fragments, of immunoglobulins,
S32
Fibrous proteins, of proteins, S24
FISH (fluorescent in situ hybridiza-
tion), S51
Fluorescent in situ hybridization,
S51
Fluorescent optical imaging, S41,
S75–S76
18
2-[ F]Fluoro-2-deoxy-D-glucose,
for PET, S71–S72
18
[ F]Fluoroethylcholine, for PET,
S72
[18F]Fluoroethylspiperone, for PET,
S73
[18F]Fluoromethylcholine, for PET,
S72
Folding, protein, S29 –S30
Frame shift mutations, S6
Functional genomics, S53–S55
 Academic Radiology, Vol 11, Suppl 1, November 2004
G Herpes simplex virus thymidine
Gadolinium, for MRI, S73–S74
Ganciclovir, radiolabeled, for PET/
SPECT, S73
Gel electrophoresis, S37–S38
in proteomics, S61–S62, S66
two-dimensional polyacrylamide,
S62, S66
Gene(s)
in DNA, S4
splicing of, S7, S18
Gene expression. See Transcription
GeneChip, in microarray analysis,
S54 –S55
Genetic Information Nondiscrimi-
nation Act of 2003, S58
Genetic mapping, S50 –S51
Genome
basic organization of, S49
of eukaryotes, S5–S6
human, S1, S49, S51, S52, S60
of prokaryotes, S5–S6
Genomics, S48 –S59
bioinformatics in, S55–S57
comparative, S52–S53
functional, S53–S55
future of, S57–S59
health benefits and, S58
social implications of, S58
structural, S48 –S51, S65–S67
translational, S57
Globular proteins, of proteins, S24
Glutamine-rich domains, in tran-
scription factors, S16
Glycosylphosphatidylinositol, in
protein synthesis, S29
Golgi membrane, protein synthesis
and, S28 –S29
Guanine, in DNA structure, S2,
S4 –S5
H M
Hairpin, RNA, S16
Heavy chains, of immunoglobulins,
S32–S33
Helix-turn-helix domain, in tran-
scription factors, S15
Hepatocyte nuclear factor, S12
kinase gene, S41, S73
Heterochromatin, S6
High-performance liquid chroma-
tography, in proteomics,
S62–S63
Histones, S5
Holoenzymes, in transcription,
S13–S14, S16
Holography, optical, S85
Homeobox, in transcription, S15
Homeodomain, in transcription,
S15
Homologous recombination, S54
Hox genes, in transcription, S15
HPCL (high-performance liquid
chromatography), in pro-
teomics, S62–S63
HSVI-tk gene, S41, S73
Human Genome Project, S1, S49,
S51, S52, S60
Human immunodeficiency virus,
RNA in, S10
Human Proteome Organization,
S61
Humanized antibodies, S33
Hybridoma technology, S33
Hypervariable regions, of immuno-
globulins, S33
I
IF initiation factors, in translation,
S26
IMAGE (Integrated Molecular
Analysis of Genomes and
their Expression), S55–S56
Immunoglobulin(s), S32–S33
Indocyanine green, for optical im-
aging, S75
Induced mutations, S6
Information management
in genomics, S55–S57
in proteomics, S67–S68
Infrared imaging, direct, S87
Initiation, of protein synthesis,
S25–S27
Insertions, in mutation, S6
SUBJECT INDEX VOLUME 11
Integrated Molecular Analysis of
Genomes and their Expres-
sion (IMAGE), S55–S56
Integrins, for PET, S72–S73, S77–
S78
International HapMap project,
S57–S58
Internet, proteomic information on,
S67–S68
Introns, S7, S18 –S19
Iron oxide, in molecular probes,
for MRI, S74
L
Lac operon, S7, S16
Lac repressor, in transcription, S15
Lagging strand, in DNA replica-
tion, S8
Lasers, in optical imaging, S85
Leading strand, in DNA replica-
tion, S7–S8
Leucine zipper, in transcription
factors, S15–S16
Leukemia, chronic myelogenous,
genomics of, S57
Libraries, DNA, S46 –S47
Licensing factors, in DNA replica-
tion, S8
Light chains, of immunoglobulins,
S32–S33
Light microscopy, S85
Liquid chromatography
high-performance, S62–S63
with mass spectrometry, S64
Lowry assay, for protein analysis,
S31
Luciferase genes, S41
Lymphoma, genomics of, S57
Lysosomes, protein degradation in,
S34
Magnetic resonance imaging
applications of, S88
in disease detection, S80 –S81
equipment for, S87–S88
molecular probes for, S73–S74, S76 –
S78
S103
 SUBJECT INDEX VOLUME 11
MALDI (matrix-assisted laser de-
sorption/ionization), in mass
spectrometry, S62–S63, S66
Mapping
genetic, S50 –S51
physical, S50 –S51
Mass spectrometry
in proteomics, S62–S64
surface-enhanced laser desorption/
ionization, S69
Matrix-assisted laser desorption/
ionization, in mass spec-
trometry, S62–S63, S66
MCM (minichromosome mainte-
nance) proteins, S8
Messenger RNA. See RNA, messen-
ger
Metabalomics, S80
Methodology, S36 –S47. See also
specific methods
bacterial transformation, S39, S41
cDNA synthesis, S46 –S47
computed tomography, S82
DNA detection, S44
DNA isolation, S36 –S37
DNA sequencing, S42–S44
electron microscopy, S65, S67
electrophoresis, S37–S38, S61–S62,
S66
high-performance liquid chromatogra-
phy, S62–S63
magnetic resonance imaging. See
Magnetic resonance imaging
mass spectrometry, S63–S65
measurements for, S47
molecular cloning, S37–S39
nuclear magnetic resonance, S65
optical imaging, S41–S42, S75–S76,
S85–S87
positron emission tomography. See
Positron emission tomography
protein functional assays, S67
radioisotope imaging, S82–S84
reporter-probe systems in, S41–S42.
See also Molecular probes
restriction enzymes, S37
RNA detection, S44 –S46
separation technologies, S37–S38,
S61–S63
S104
Academic Radiology, Vol 11, Suppl 1, November 2004
single photon emission computed
tomography, S71–S73, S77–
S78, S83
supplies for, S47
transfection, S41
ultrasonography, S74 –S75, S78
x-ray crystallogaphy, S65
7-Methylguanosine, in post tran-
scriptional processing, S18
Micro RNA, S12–S13
Microarray technology, S54 –S55,
S67
Microbubbles, for ultrasonography,
S74 –S75, S78, S84
Microsatellites, S50
Microscopy
electron, S65
light, S85
Minichromosome maintenance
(MCM) proteins, S8
Minisatellites, S50
miRNA (micro RNA), S12–S13
Molecular cloning, S37–S39
Molecular imaging
definition of, S1, S80
equipment for. See Equipment, for
molecular imaging
practice of, S91–S94
stage 1 (see morphology, think
molecular biology), S91–S92
stage 2 (combine imaging informa-
tion with molecular diagnostic
information), S92
stage 3 (obtain molecular informa-
tion using imaging), S92–S93
stage 4 (personalize treatment us-
ing combined molecular imag-
ing and therapy), S93
tasks of, S80 –S81
Molecular probes, S71–S78
development of, S77–S78
for magnetic resonance imaging,
S73–S74, S76 –S78
for multimodality imaging, S76 –S77
for optical imaging, S75–S76
for positron emission tomography,
S71–S73, S77–S78
for single photon emission computed
tomography, S71–S73, S77–
S78, S83
for ultrasonography, S74 –S75, S78
Monoclonal antibodies, S33
MRI. See Magnetic resonance imag-
ing
mRNA. See RNA, messenger
Multimodality imaging, molecular
probes for, S76 –S77
Mutagens, definition of, S6
Mutations, types of, S6
N
Near-infrared region, fluorescent
probes emitting in, S75–S76
Ninhydrin reaction, for protein
analysis, S30 –S31
Nonpolar amino acids, S23
Northern blotting, in RNA detec-
tion, S44
Nuclear isotope imaging, S82–S84
Nuclear localization signal, in
translocation, S28
Nuclear magnetic resonance, in
proteomics, S30, S65
Nucleosomes, S5
Nucleotides, in DNA, S2, S4 –S5
O
Octreotide, radiolabeled, for PET,
S72
Okazaki fragment, in DNA replica-
tion, S8
Oligochips, in microarray analysis,
S54 –S55
Oligomeric proteins, S24
Open reading frame analysis, S53–
S54
Operons, S6 –S7, S16
Optical coherence tomography, S87
Optical imaging, S41–S42
applications of, S87
equipment for, S85–S87
molecular probes for, S75–S76
ORF (open reading frame analy-
sis), S53–S54
Ovarian cancer, proteomics tech-
niques in, S69
 Academic Radiology, Vol 11, Suppl 1, November 2004
P equipment for, S83–S84
PAGE (polyacrylamide gel electro-
phoresis), two-dimensional,
S62, S66
Paramagnetic molecular probes,
for MRI, S73–S74
Pax genes, in transcription, S15
Penciclovir, radiolabeled, for PET/
SPECT, S73
Peptide(s)
mass mapping of, in mass spectrome-
try, S63–S64
post-translational processing of, S27–
S32
solid-phase synthesis of, S32
Peptide bonds, in amino acids, S24
Peptidyl transferase, in protein
synthesis, S27
Perfluorocarbons, for ultrasonogra-
phy, S75
PET (positron emission tomogra-
phy), S41–S42
computed tomography with, S82
equipment for, S83–S84
molecular probes for, S71–S73, S77–
S78
Phage display, for antibody pro-
duction, S33
Phenomics, S57
Phosphodiester linkages, in DNA,
S5
Physical mapping, S50 –S51
Plasmid(s), cloning of, S37, S39
Plasmid DNA, isolation of, S36 –
S37
Point mutations, S6
Polar amino acids, S23
Poly (A) polymerase, in transcrip-
tion termination, S18
Polycistronic mRNA, S7
Polymerase chain reaction, S42
real-time, S46
reverse transcription, S44
Polymorphisms, S49 –S50
Polypeptides, definition of, S22
Positron emission tomography,
S41–S42
computed tomography with, S82
molecular probes for, S71–S73, S77–
S78
Pribnow box, in transcription, S14,
S16
Primary structure, of proteins, S24
Primary transcripts, S12
Primase, in DNA replication, S8
Probes, molecular. See Molecular
probes
Prokaryotes
gene structure in, S6 –S7
genome of, S5–S6
ribosomal RNA in, S12
transcription in, S16
translation in, S24 –S27
Proline-rich domains, in transcrip-
tion factors, S16
Promoters, in transcription, S14
Proof-reading, in DNA replication,
S7
Prosthetic group, of proteins, S24
Proteasome complexes, S34
Protein(s), S22–S35. See also spe-
cific proteins
analysis of, S30 –S31
conjugated, S24
definition of, S22
degradation of, S33–S34
fibrous, S24
globular, S24
number of, S22
oligomeric, S24
on organism scale. See Proteomics
peptide sequencing in, S31–S32
simple, S24
structures of, S24, S30
synthesis of
amino acid building blocks for,
S22–S24
elongation in, S27
folding in, S29 –S30
initiation of, S25–S26
peptide post-translational process-
ing in, S27–S32
ribosomes in, S24
solid-phase, S32
termination of, S27
transcription in. See Transcription
SUBJECT INDEX VOLUME 11
translation accessory factors in,
S25
translation in, S24 –S27
translocation in, S27–S29
tRNA in, S24 –S25
Protein pattern diagnostics, S69
Proteomics, S60 –S70
bioinformatics and, S67–S68
challenges to, S61
clinical, S68 –S69
current studies in, S61
definition of, S60
imaging applications of, S69
techniques for, S61–S67
electron microscopy, S65, S67
functional assays, S67
mass spectrometry, S63–S65
nuclear magnetic resonance, S65
separation, S61–S63
structural genomics, S65–S67
x-ray crystallography, S65
Proximal elements, in transcrip-
tion, S14
Purines, in DNA, S2, S4 –S5
Pyrimidines, in DNA, S2, S4 –S5
Q
Quantum dots, for optical imaging,
S75, S86
Quaternary structure, of proteins,
S24
R
Radioisotope imaging, S82–S84
Real-time polymerase chain reac-
tion, S46
Release factors, in translation, S27
Replication, DNA, S7–S8
Reporter probe systems, S41–S42
Repressor elements and sequences,
in transcription, S14, S15
Restriction enzymes, S37
Restriction fragment length poly-
morphisms, S51
Restriction mapping, S51
Reverse transcriptase, action of,
S10
S105
 SUBJECT INDEX VOLUME 11 Academic Radiology, Vol 11, Suppl 1, November 2004

Reverse transcription polymerase RNA interference, S54 Single nucleotide polymorphisms,

chain reaction, in RNA de- RNA polymerases, in transcription, S50
tection, S44 S11, S13–S14, S16 –S20 Single photon emission computed
RFLPs (restriction fragment length RNA primer, in DNA replication, tomography, molecular
polymorphisms), S51 S8 probes for, S71–S73, S77–
Rho factor, in transcription, S16 RNAPs. See RNA polymerases S78, S83
Ribonucleic acid. See RNA Rough endoplasmic reticulum, pro- siRNA (short interfering RNA),
Ribonucleotides, S5 tein synthesis and, S28 S12–S13
Ribosomal RNA. See RNA, ribo- rRNA. See RNA, ribosomal Small interfering RNAs, S54
somal Small nuclear RNA, S12
Ribosomes, S12 S Small nucleolar RNA, S12
in translation, S24 Sanger method Small temporal RNA, S12–S13
in translocation, S27–S28 for DNA sequencing, S42–S43 Smart molecular probes, S75–S76
RNA for peptide sequencing, S31–S32 Smooth endoplasmic reticulum,
detection of, S44 –S47 Scanning model, in translation, S26 lipid synthesis and, S28
discovery of, S10 Secondary structure, of proteins, snoRNA (small nucleolar RNA),
generation of, S13. See also Tran- S24 S12
scription SELDI-MS (surface-enhanced laser SNPs (simple sequence length poly-
hairpin, S16 desorption/ionization mass morphisms), S50
isolation of, S44 spectrometry), S69 snRNA (small nuclear RNA), S12
messenger, S6, S12 Selectins, in molecular probes, for Social implications, of genomic re-
formation of, S18 –S20, S27 MRI, S74 search, S58
structure of, S10 Semi-conservative DNA replication, Solid-phase peptide synthesis, S32
micro, S12–S13 S7 Southern blotting, in DNA detec-
post transcriptional processing of, Sense strand, in RNA replication, tion, S44
S18 –S20 S13 Spatial resolution, in molecular
pre-messenger, S18 –S20 Sensitivity and specificity, of molec- imaging, S81
quantification of, S44 ular imaging methods, S81 SPECT (single photon emission
replication of, S13 Separation technologies, S37–S38 computed tomography), mo-
ribosomal, S6, S12 Sequence tagged site mapping, S51 lecular probes for, S71–S73,
5.8S, S12 Sequencing S77–S78, S83
5S, S12 DNA, S42–S44 Spectrometry, mass, in proteomics,
16S, S12 genome, S51 S62–S64, S69
18S, S12 peptide, S31–S32 Spliceosomes, S12, S18
23S, S12 Shine-Dalgarno sequence, in trans- Splicing, gene, S7, S18
28S, S12 lation, S26 Spontaneous mutations, S6
gene for, S20 Short interfering RNA, S12–S13 SSLPs (simple sequence length
in translation, S24 Sickle cell disease, protein misfold- polymorphisms), S50
short interfering, S12–S13 ing, S30 Stains, for electrophoresis, S61
small interfering, S54 Sigma factor, in transcription, S16 Start codons, S26
small nuclear, S12 Signal recognition particle, in Stop codons, S27
small nucleolar, S12 translocation, S28 stRNA (small temporal RNA), S12–
small temporal, S12–S13 Signal-to-noise ratio, in molecular S13
structure of, S5, S10 –S11 imaging, S81 STRs (microsatellites), S50
transfer, S6, S12 Silver stain, for gel electrophoresis, Structural genomics, S48 –S51
structure of, S24 –S25 S61 genetic mapping in, S50 –S51
in translation, S24 –S25 Simple proteins, S24 genome sequencing in, S51
in viruses, S10 Simple sequence length polymor- physical mapping in, S50 –S51
Xist, S13 phisms, S50 polymorphisms in, S49 –S50

S106
 Academic Radiology, Vol 11, Suppl 1, November 2004
techniques for, S65–S67
STS (sequence tagged site map-
ping), S51
Superparamagnetic probes, for
MRI, S73–S74
Surface-enhanced laser desorption/
ionization mass spectrome-
try, S69
SWISS-PROT data base, S68
T Transfection, of eukaryotic cells,
TaqMan method, for polymerase
chain reaction, S46
TATA box, in transcription, S14,
S17
TATA-less promoters, in transcrip-
tion, S14
Telomeres, S6
Template strand, in RNA replica-
tion, S13
Temporal resolution, in molecular
imaging, S81
Termination, in RNA replication,
S13, S18
Tertiary structure, of proteins, S24
TETA-octreotide, for PET, S72
TFII transcription factors, S17
Thymidine, radiolabeled, for PET/
SPECT, S73
Thymine, in DNA structure, S2,
S4 –S5
Transcription, S10 –S21
definition of, S10, S13
DNA elements in, S14 –S15
in eukaryotes, S15–S18
process of, S13
processing after, S18 –S20
in prokaryotes, S16
reverse, S10
RNA in. See RNA
RNA polymerases in, S13–S14
splicing after, S7, S18
termination signals in, S13, S18
Transcription activation domains,
in transcription factors, S16
Transcription factors, in eu-
karyotes, S15–S18
Transcriptional activation domain,
of transcription factors, S15
S41
Transfer RNA. See RNA, transfer
Transformation, bacterial, S39, S41
Transgene expression, imaging of,
S73
Transition, in mutation, S6
Translation
accessory factors for, S25
definition of, S6, S10
elongation of, S27
initiation of, S26 –S27
ribosomes in, S24
termination of, S27
tRNA in, S24 –S25
Translational genomics, S57
Translocation, protein, S27–S29
Transversion, in mutation, S6
Triplet genetic code, S6
tRNA (transfer RNA). See RNA,
transfer
Trp repressor, in transcription, S15
Two-dimensional polyacrylamide
gel electrophoresis, in pro-
teomics, S62, S66
U
Ubiquitin-dependent proteolytic
systems, S34
SUBJECT INDEX VOLUME 11
Ultrasonography
equipment for, S84 –S85
molecular probes for, S74 –S75, S78
Upstream regulatory sequences, in
transcription, S14
Urokinase plasminogen activator,
as molecular probe, S76
V
Variable region, of immunoglobu-
lins, S33
Vectors, for molecular cloning, S37,
S39
VNTRs (minisatellites), S50
W
Watson, J.D., DNA discovery by,
S4
Western blotting, in protein sepa-
ration, S61–S62
Wilms’ tumor gene, mutations in,
S15
Wobble hypothesis, in translation,
S25
WT1 gene, mutations of, S15
X
X chromosome, silencing of, S13
Xist RNA, S13
X-ray, in molecular imaging, S82
X-ray crystallography, in proteom-
ics, S65
Y
Yeast two-hybrid system, in pro-
teomics, S67
Z
Zinc finger domains, in transcrip-
tion factors, S15
S107
Author Index

Azok, J.T., S60 Li, K.C.P., S1, S10, S22, S36, S48, Thomasson, D.M., S79
Bednarski, M., S1 S60, S71, S79, S91 Vuu, K., S48
Danthi, S.N., S71 Pandit, S.D., S1, S10, S22, S36, S48,
Gharib, A., S79 S60, S71

S108