Concepts and Techniques in Molecular Biology
William P. Sheffield
I REALIZED that molecular biological concepts
and techniques were not always embraced with
enthusiasm at the first meeting I attended o f the
American Society of Hematology in Boston in
1990. At that meeting, a plenary speaker convulsed
his audience with laughter by telling a joke. The
punchline involved a hematologist and an internist
who jumped out of an airplane without parachutes
rather than listen to a molecular biologist rhapso-
dize about, gene structure. Nevertheless, it is even
more difficult today than it was 7 years ago to pick
up a copy of Blood or Transfusion, for example,
without coming across numerous articles in which
molecular biological approaches are use d . This
article will provide an overview aimed at demysti-
fying these now widely used approaches.
Molecular biology can be thought of as a disci-
pline lying at the confluence of biochemistry,
genetics, microbiology, and protein chemistry. Con-
ceptually, molecular biology focuses on nucleic
acids and the protein products they encode; opera-
tionally, all molecular biological experimentation
involves deoxyribonucleic acid (DNA) in one way
or another, and specifically the manipulated DNA
molecules known as recombinant DNA.
DNA is the biomolecule that contains all genetic
information. Uncertainty over what material com-
prised the gene ended in the late 1940s and the
early 1950s when it was shown that purified
preparations of DNA transferred pathogenicity be-
tween bacterial strains, l and that viral DNA, not
viral coat protein, enters cells and directs viral
propagation? Radiograph diffraction patterns of
DNA crystals showed a helical structure. The
realization that the diameter of crystalline DNA
was too great for a single strand led Watson and
Crick to propose that DNA is a double-stranded
helix)
As shown in Figure 1, each DNA strand is a
polymer of substituted sugars joined by phosphate
linkages between the 5' carbon of one deoxyribose
and the 3' carbon of the next. The substitutions
(attached to the 1' carbon) are purine (adenine, A,
or guanine, G) or pyrimidine (cytosine, C, or
thymine, T) bases. The two strands are antiparallel,
in that one lies 5' to 3', whereas the other lies 3' to
5', and complementary, in that A on one st{and
always binds T on the other (via two hydrogen
Transfusion Medicine Reviews, Vol 11, No 3 (July), 1997: pp 209-223
An Overview
bonds), whereas C on one strand always binds G on
the other (via three hydrogen bonds). The sugar or
phosphate backbone forms the long axis of the
molecule, with the nitrogenous bases inside and
oriented perpendicular to the backbone. The mono-
meric unit that serves as a building block for DNA
polymers comprises a base-substituted deoxyribose
sugar attached to three phosphate groups, and is
known as a deoxynucleotide triphosphate.
Understanding the structure of the double helix
set the stage for our current understanding of how
the genetic information held in DNA is propagated
to ensure survival of daughter cells, and transcribed
and translated to form the protein products that do
the work of the cell.
THE FLOW OF GENETIC INFORMATION
DNA, as Information Repository
The human genome, or the sum total of all DNA
in a human somatic cell, is approximately 3 billion
base pairs (bp). 4 In other words, if all human genes
and intergenic DNA were arranged on a single
DNA molecule, it would constitute a double helix
with 3 billion substituted deoxyribose units on each
strand. In reality, the human genome is divided
among 23 chromosomes. Somatic cells contain 46
chromosomes (ie, 6 billion bp), having obtained 23
chromosomes from each parent in the union of
sperm and egg. Each chromosome is a single,
linear, double-stranded DNA molecule containing
thousands of genes. When cells grow and divide,
both daughter cells must receive a full complement
of cellular DNA. In a process known as replication,
processive enzymes called polymerases use each
DNA strand as a template for the synthesis of a new
From the Department of Pathology, McMaster University,
Hamilton, Ontario, Canada, and Canadian Red Cross Society
Blood Services. WPS is a Bayer~Canadian Red Cross Society/
Medical Research Council of Canada Scholar.
Supported by the Canadian Red Cross Society Research and
Development Fund, and the Heart and Stroke Foundations of
Canada and Ontario.
Address reprint requests to William P. Sheffield, PhD, Associ-
ate Professor, Department of Pathology, HSC 3NIO, McMaster
University, 1200 Main St West, Hamilton, Ontario, Canada L8N
3Z5.
Copyright 9 1997 by W.B. Saunders Company
0887- 7963/97/1103-000653. 00/0
209
210
:iparallel strands
fJ Hydr'~ogen" /2,_c a,c~~
Complementarity
Fig 1. Structure of the DNA double helix. The upper arrows
indicate the schematic opening of the double helix to show
the antiparallel arrangement of the deoxyribose backbone.
The positions of the four carbons within the ring and the
single carbon outside the ring structure are indicated and
numbered (1-5'-C). Phosphate groups are indicated diagramati-
cally by a circled P, and the purine or pyrimidine bases are
shown as a boxed B. The lower arrow indicates an exploded
schematic diagram of the base interactions, showing A:T and
G:C complementary base pairing.
complementary strand (using only the 5' to 3'
direction). Each daughter cell thus receives one
"old" strand of DNA and one newly synthesized
complementary strand for every helix in this semi-
conservative process.
Given that the protein products of genes are
made up of combinations of twenty different amino
acids, it is not immediately obvious how DNA,
with only four different subunits, can encode all
this information. This became clear when it was
learned that the molecular machinery that recog-
nizes the genetic code reads, or recognizes, three
bases at a time. The number of different ways that
four bases can be arranged in codons, or groups of
three, is 4 3 , o r 64, providing more than sufficient
means to encode 20 amino acids. 5
As might be expected, the genome is not simply
a continuous stretch of coding information. A
human gene contains both coding and non-coding
DNA. Non-coding DNA includes genetic control
WILLIAM P. SHEFFIELD
regions found primarily 5', or "upstream" of the
coding regions, intervening sequences that inter-
rupt coding regions, and intergenic and repetitive
DNA elements of unknown function. Coding DNA
blocks are called exons and the non-coding DNA
that interrupts exons are termed introns. The func-
tion of introns, which are absent from bacterial
DNA, is unclear, but they may play an important
evolutionary role by allowing greater ease of
joining together exons to form new proteins in
higher organisms. 6 -~
In terms of cellular localization, the vast major-
ity of human DNA is found in the nucleus. A small
circular DNA molecule is found within mitochon-
dria, composed of 16,569 bp, which encodes sub-
units of mitochondrial proteins important in energy
metabolism. 7 Nuclear DNA is found complexed to
proteins such as histones in a nucleoprotein com-
plex that includes ordered structures called nucleo-
somes, in which DNA is made compact by folding
around the protein scaffold.8 Extranucleosomal
stretches of DNA are able to bind other, less
abundant proteins that pave the way for transcrip-
tion of the DNA message into an intermediary
messenger, a related nucleic acid called RNA
(ribonucleic acid).
R N A as a Genetic Messenger
RNA differs from DNA in that it is single-
stranded and its sugar backbone contains a hy-
droxyl group on the 2' carbon, making it ribose
rather than deoxyribose. Like DNA, each mono-
meric unit is substituted with one of four purine or
pyrimidine bases, but RNA uses uracil (U) instead
of thymine (T). The process of transferring genetic
information from DNA to RNA, transcription, is
mediated by RNA polymerases. 9 These enzymes
use one strand of the DNA as a template, and
produce an RNA chain in which every 5'-ACGT-3'
of DNA produces an antiparallel 5'-ACGU-3'
,RNAtranscript; because transcription, like replica-
tion, proceeds in the 5' to 3' direction, this means
that the RNA polymerase actually uses the non-
coding strand of DNA as a template. In any
chromosome, which strand is coding and which is
non-coding varies for different genes. RNA tran-
scripts that encode proteins are known as messen-
ger' RNAs (mRNAs) and are generated by RNA
polymerase II, whereas RNA polymerase I gener-
ates ribosomal RNAs that play essential roles in the
ribosome, .and RNA polymerase III generates both
MOLECULAR BIOLOGY OVERVIEW
transfer RNAs (tRNAs, adaptor molecules that
bring amino acids to the RNA template in transla-
tion) and another rRNA.
As shown schematically in Figure 2, the initial
mRNA transcript of a nuclear gene is processed in
three ways: its 5' terminal phosphate is "capped"
by bonding to the 5' carbon of a 7-methylated
guanine; a stretch of adenosine residues are added
to its 3' end to form a poty(A) tail; and portions of
the transcript corresponding to introns are spliced
out. These modifications take place in the nucleus,
and are mediated by various enzymes and com-
plexes that recognize signals in the nascent tran-
script. 1~After processing, the mature mRNA enters
the cytoplasm through nuclear pores. Although
A eukaryotic mRNA and processes along the mRNA
( GENE > in the 5' to 3' direction. When the sequence AUG is
promoter
I I
o: , : oO
RNA Pol II Transcription
m I
Processing
mTG ~ 'AAAAA n
Splicing
mTG ~ A A A A A n
~ Exportfrom nucleus to cytoplasm
B AUG UGA
mTG I U ....... I AAAAAn
5'-UTR ORF 3'-UTR
Fig 2. Transcription of eukaryotic genes. (A) shows the
transfer of genetic information from a hypothetical three-exon
gene. After transcription by RNA polymerase II (RNA Pol II), a
primary RNA transcript containing the transcribed introns is
formed. This primary transcript is processed by the addition of
a 5' cap structure (m7G) and a 3' polyadenylated tract
(AAAAn). The processed mRNA is then spliced to remove the
transcribed introns, yielding the mature mRNA, which is then
exported from the nucleus to the cytoplasm. (B) shows the
same mature mRNA whose generation is detailed in A, but
with additional information. The position of the start IAUG)
and stop (UGA) is shown in the upper line diagram. These
points serve to define the open reading frame (ORF), and the
5'-untranslated region (5'-UTR) and 3'-untranslated region
(3'-UTR) t h a t f o l l o w it, as shown in the lower line diagram.
211
usually greatly reduced in size compared with the
primary transcript, the mature mRNA still contains
additional non-coding information, in two zones: at
the 5' end, referred to as the 5' untranslated region
(5' UTR, generally of 50-150 bp); and at the 3' end,
referred to as the 3' untranslated region (Y-UTR,
generally Of 200-1000) bp. The 3'-UTR is thought
to contain sequences that can contribute to the
determination of mRNA stability.
Translated Protein As "Final" Product
RNA translation takes place in the cytoplasm
when mature mRNAs bind ribosomes. Eukaryotic
ribosomes 0e, those from organisms with nuclei)
are made up of 40S and 60S ribosomal subunits,
each of which are rRNA-protein complexes, u The
small subunit attaches to the cap structure of
encountered, a methionine-can'ying tRNA binds to
the codon, and recruits the other 60S ribosomal
subunit to form the full 80S ribosome, with the aid
of initiation factor proteins. The ribosome then
moves along the mRNA template, reading codons
in the reading frame, or sequence, determined by
the placement of the AUG. As shown schematically
in Figure 3, as the next aminoacyl tRNA binds the
second codon, it "steals" the methionine from the
first tRNA, forming a peptide bond, and moves
from the A (aminoacyl) ribosomal site to the P
(peptidyl) site, forcing ejection of the deacylated
tRNA that formerly was charged with methionine.
The nascent polypeptide chain is therefore built in
the amino to carboxyl direction. Chain elongation
continues, until UGA, UAG, or UAA termination
signals are encountered. Because translation is
processive, and because the ribosome sterically
covers only about 80 nucleotides, many ribosomes
can be in the process of translating even a small
mRNA at the same time) 2
With chain termination and release of the pep-
tide, protein synthesis is complete, at least from the
point of view of having completed the task of
assembling the linear chain of amino acids. How-
ever, for proteins to achieve their full biological
activity, they must be maintained in an appropriate
conformation, and reside in the appropriate cellular
environment) 3 This is achieved by a combination
of spontaneous folding, folding and disulphide
bond formation assisted by protein chaperones, and
post- or co-translational modification. The later
 212 WILLIAM P.SHEFFIELD

REGULATING GENE EXPRESSION

Every cell in the body contains the same comple-
Amino-acyl
tRNA binding ment of DNA, arranged in the same way. 14 The
only exceptions are cells like committed B-
lymphocytes, in which a specific rearrangement of
5' 3'
mRNA the immunoglobulin locus has taken place; sex
cells, which are haploid; and unusual specialized
"cells" like erthyrocytes and platelets that have lost
their DNA during maturation. In spite of the
identity of genetic information found in all other
cells, different cell types exhibit vastly different
~ Peptidebond
formation patterns of gene expression, in that genes that are
practically silent in one cell type can be abundantly
expressed in others. How is this control achieved?
5' 3'
Although the molecular details of this essential
question are under continuous, and vigorous inves-
tigation, much of the broad outlines of the answer
have become apparent. Enkaryotic genes are pre-
dominantly regulated at the level of transcriptioN,
however in some cases post-transcriptional control
( ~ ~ Lossof uncharged is an important additional influence. Although gene
tRNAand amino-
acyl tRNAbinding regulation involves both DNA structures and the

5'

Fig 3. Translation of a eukaryotic messenger RNA by a Rever e

ribosome. This schematic diagram shows the eukaryotic 80S transcription~ Transcription
ribosome (RIBOSOME), highlighting its 5OS and 30 S subunits
(su). The exit (E),peptidyl-tRNA (P), and amino-acyl tRNA (A)
binding sites are shown, as well as three ribonucleotides ~ [E~>E~ [E~>RNAsN~176
iNNN) preceding the start codon (AUG), and a Phe codon
(UUU), on the template (mRNA). The upper figure shows a
Met-tRNAbound via its anticodon to the AUGcodon,with an
incoming Phe-acyl-tRNA. The middle diagram shows peptide Tra'nslation
bond formation, and transfer of t h e growing peptide chain to
the former Phe-tRNA.The lower diagram shows exit of the
former Met-tRNA, movement of the ribosome along the
mRNAto transfer the nascent chain-tRNA to the P site, and an
incoming tRNA corresponding to the third codon.
Sorting
Folding
Post-translational
modifications often involve the interaction of ad- Modifications
dressing signals on the nascent polypeptide with
Protein (with
translocation proteins on different organelles, sig- full biological
nals that can be removed by proteolysis after, or activity)
during, protein translocation. Finally, proteins are
often modified by the covalent addition of addi- Fig 4. Flow of genetic information. The flow of. genetic
tional chemical structures, eg, N- or O-linked sugar information from DNA to RNA to prQtr isr s~hematically
chains, or phosphate or acyl groups. Once again, shown, with the distinction being made between protein
product, and protein product that has been appropriately
these modifications occur in response to signal folded and post-translationally modified and delivered to its
sequences carried within the amino acid sequence appropriate location. The interrupted arrowsindicate the less
of the protein, and complete the flow of genetic commonly encountered flow of information from RNAto DNA
(reverse transcription, often by retroviruses) and the impor-
information from DNA to fully functional protein tant but less numerous RNA products that do not encode
product (Figure 4). protein produ.cts(eg,ribosomal and transfer RNAs).
MOLECULAR BIOLOGY OVERVIEW
regulatory proteins that bind these structures, act-
ing in concert, the control regions and the control
factors will first be examined separately. DNA
sequences lying on the same chromosome as the
gene under consideration are referred to as "cis-
acting sequences," whereas the transcription fac-
tors, often encoded on another chromosome, are
referred to as "trans-acting factors." 15
Cis-acting Sequences
A promoter is a region of DNA essential to gene
expression. This gene control region lies immedi-
ately 5' tothe initiation of transcription of a given
gene, and usually comprises no more than 100 to
200 base pairs. The promoter serves to recruit and
guide RNA polymerase molecules to initiate tran-
scription at the appropriate site. This guidance is
indirect, because the general transcription factors
which bind to promoter elements themselves form
a multi-protein pre-initiation complex with RNA
polymerase. These factors are proteins named TF
(transcription factor) followed by the number of the
RNA polymerase with which they interact, and a
letter.16
Smaller functional modules exist within the
promoter region. Taking the first transcribed base
as + 1, and defining upstream bases as negatively
numbered, many promoters contain a DNA se-
quence motif called the TATA box between - 2 5
and - 3 5 . TFIID commences the mRNA transcrip-
tion initiation process by binding to the TATA box,
assuring an appropriate start site and unidirectional-
ity. 17 At least five other factors and RNA polymer-
ase then bind to form the pre-initiation complex,
which is capable of low-level, basal transcription.
Stronger promoter activity requires upstream acti-
vator sequences such as the CCAAT and GC boxes,
which are often found between - 7 0 and - t 0 0 ;
unlike the TATA box, the latter two elements can be
positioned on the non-coding strand. Even greater
increases in transcriptional activation are mediated
by cis-acting sequences which are usually located
further upstream.
The upstream activator sequences act by binding
transcription factors that usually are bimodal in that
they combine a DNA-binding site with a site
capable of binding a component of the pre-
initiation complex. As such, these activators re-
semble enhancer elements in the means by which
they increase basal transcription. Enhancers differ
from promoters in that they can act at great
213
distances from the start of transcription, and are
position- and orientation-independent. In addition,
enhancer elements cannot enhance transcription in
the absence of a promoter. Enhancer-binding pro-
tein transcription factors are thought to overcome
their seemingly distant location through looping of
the DNA, which brings them into sufficient proxim-
ity to their target gene to activate transcription. 18
Similar elements that operate to prevent or down-
regulate transcription are called silencers.
An important point is that most genes contain
multiple binding sites for both ubiquitous and
tissue- or temporally-specific transcription factors.
This provides for part of the diversity of gene
expression patterns; other contributions arise from
the nature of the trans-acting factors themselves. 19
Trans-acting Factors
As discussed above, transacting factors are DNA
binding proteins whose binding to DNA results in
activation of transcription. Most commonly, this
end is achieved by the binding of other transcrip-
tion factors by the trans-acting factor, with the
other factors either being associated with or a direct
part of the basal transcriptional machinery. These
complex interactions between numerous DNA mo-
tifs, DNA binding proteins, and transcription factor-
binding proteins make possible myriad different
patterns of gene expression, without the energeti-
cally unfavourable need for myriad individual
transcription factors. Further parsimony is reflected
in the organization of transactivators into structur-
ally related groups, and in transactivators that
function as heterodimers, Common structural mo-
tifs seen in transactivating proteins include the zinc
finger, leucine zipper, and helix-turn-helix (HTH)
domains.
The zinc finger is a cormnon structural motif
present in transactivators, consisting of a 30 amino
acid zone containing two cysteines and two histi-
dine residues that coordinately bind a zinc ion. 2~
This extended conformer wraps part way around
the double helix, contacting both a three base-pair
subsite and the sugar/phosphate backbone. Hun-
dreds of proteins contain zinc finger domains. Zinc
binding is also seen in the nuclear receptor super-
family of hormone binding proteins, in this case
using two cysteines and two histidines. 21 These
dimeric proteins bind diffusible hormones like
estrogen or glucocorticoids in a step that unmasks
their DNA binding domains. Following transport to
214
the nucleus, they then transactivate target genes
containing a cis-acting hormone response element.
Leucine zipper trans-acting factors also form
dimeric units whose subunits intertwine via c~-heli-
cal coil-coil interactions. 22 Examples of leucine
zipper (or bZIP for basic zipper) proteins include
the cyclic AMP response element binding protein
(CREB). Regulated phosporylation of CREB mono-
mers promotes dimerization and transactivation. 23
Heterodimers also can form between different
proteins containing leucine zipper domains, such as
the jun and fos proteins that comprise the AP-1
transactivator. 24 Multimerization domains are also
found in HTH proteins such as octomer-binding
proteins, the pituitary-specific Pitl factor, and devel-
opmentally important homeodomain proteins, 25and
even more strikingly in the helix-loop-helix pro-
teins such as muscle-enriched MyoD protein, z6 By
forming heterodimers, a relatively small number of
transacting factors can generate great diversity in
regulating different genes.
Although most trans-acting factors are transcrip-
tional activators, examples exist of RNA-binding
proteins that modulate the expression of a gene at
the post-transcriptional level. For example, the
same regulatory proteins bind to stem-loop struc-
tures in the 3' end of transferrin receptor mRNA
and the 5' end of the ferritin mRNA when iron
stores are low, increasing the stability of the former
transcript and decreasing the transcription of the
latter. 27 This variation on the theme of gene regula-
tion completes our survey of how information
encoded in DNA is used to produce proteins or
regulate gene activity, and sets the stage for consid-
eration of two topics: how the flow of information
is disturbed by mutation, and how the flow of
information can be tapped by molecular biological
techniques (see next section). Although a rigourous
treatment of genetic mutation is far beyond the
scope of this review, the simple concept that things
can go wrong at any step in the transfer of
information is useful. All genetic mutations involve
DNA changes at the level of the gene, ie, insertions,
alterations, or deletions of sections of DNA of
single oa" multiple base pairs. Alterations to pro-
moter or enhancer regions can greatly reduce
transcription, whereas those affecting mRNA splic-
ing or stability can prevent or reduce gene product
formation in spite o f normal primary mRNA tran-
scription. Alterations to the coding region can shift
the reading frame such that a completely different
WILLIAM P. SHEFFIELD
amino acid sequence is translated, o1"translation is
abruptly terminated. Alternatively, single amino
acid changes can abrogate signal sequence func-
tion, preventing the gene product from reaching its
proper location, or disrupt global protein folding, or
disable a ligand or substrate binding site: If the
genetic mutation affects a key regulatory protein,
like a trans-acting factor, whole networks of genes
may fail to be appropriately transcribed (for ex-
ample, mutations in the Pit-1 transcription factor
underlie combined pituitary hormone disorders28).
TAPPING THE FLOW OF GENETIC
INFORMATION
Although the "central dogma" of molecular
biology states that genetic information flows from
gene to RNA to protein, much of the explosion of
genetic knowledge fuelled by molecular techniques
has become available through reversing or tightly
channelling that flow. The molecular biological
technical !'mind set" mixes reductionist, in vitro
experimentation with a willingness to let microor-
ganisms, cultured cells, or even whole organisms
"do the work.'"As will hopefully become clear in
this section, and as shown in Figure 5, there are
many different ways to use molecular techniques,
and many potential starting points.
Working With DNA and RNA
Experimental manipulation of both RNA and
DNA requires reasonable, but differing levels of
care. Although the protection of samples from
microbial and non-specific organic contamination
is a given in both instances, DNA is relatively
stable; inclusion, where possible, of chelating agents
(eg, ethylenedianainetetraacetic acid, EDTA) usu-
ally serves to protect DNA from endonuclease
degradation. On the other hand, its single-stranded
cousin, RNA, is susceptible to attack by resilient
and ubiquitous ribonucleases that can withstand
autoclaving. Minimizing contamination, cross-
contamination, and losses of RNA and DNA is
achieved through the extensive use of disposable
plasticware. Both nucleic acids can be concentrated
by alcohol precipitation. Whereas gentle~l~ysis pro-
cedures favour the isolation of high molecular
weight DNA that would otherwise be sheared,
rapid lysis in the presence of strong denaturing
agents is required for efficient RNA isolation. The
bases of both DNA and RNA absorb strongly at 260
nm, a property that can be used for yield determina-
MOLECULARBIOLOGYOVERVIEW
Fig 5. Flow diagram of mo-
lecular biological experimenta-
tion. Possible experimental strat-
Differential < ~ 1 ~
display
egies are schematically depicted,
starting from either native pro-
tein, RNA, or DNA (boxed). For
instance, the purified native pro-
tein can be used to raise an anti- Antibody E ~
body+The antibody probe can be ~/
used to screen a cDNA library
generated from RNA. Once se- cDNAclone
quenced, the cDNA correspond- ~ ~[~
ing to the native protein can be
used to express either native or ~' DNAs~uence<~
mutated recombinant protein, or w Exonmapping
to screen a genomic library gen- Degenerate
erated from DNA. Resulting ge-
oligonucleotides
nomic clones can be used to /~
construct vectors for the genera-
tion of knock-out mice, in which
the gene corresponding to the Transgenic
original native protein has been
mice
inactivated, and to characterize
the gene's promoter elements.
tion, with protein contamination being reflected by
comparison to absorbance at 280 nm. Although
physicochemical methods involving the ultracentri-
fugation of DNA and RNA through density gradi-
ents of cesium chloride can be used to obtain highly
purified samples, these methods have been largely
supplanted in recent years by the development of
commercial ion-exchange type resins capable of
rapid and efficient nucleic acid purification.
The large size, strong negative charge, and
similarity of substitution between monomeric sub-
units of RNA and DNA render them ideal mol-
ecules for resolution in an electric field. Electropho-
resis through horizontal gels of agarose is widely
used for separation and visualization of DNA and
RNA, with the former being easier to visualize
through the use of ethidium bromide, a fluorescent
dye that intercalates between DNA strands. Electro-
phoretic mobility of RNA that is proportional to its
size alone requires the prevention of intrachain
interactions, usually achieved by incorporation of
denaturing agents such as formaldehyde and/or
formamide. Analogous denaturation is required to
separate DNA strands through incorporation of
urea in acrylamide gels and formamide in samples.
Enzymatic Tools
Manipulation of DNA and RNA required the
development of techniques for specific cleavage,
modification, copying, joining, and controlled and
general degradation. Usually this was achieved by
215
DNAfingerprinting
[E~> Northern Southern < ~ ~
Blot Blot
cDNAlibrary Genomiclibrary
Screenlibrary Probe
~, /~+~/ Genomioclone
~'
~ DNAsequence
Mutagenesis
Heterologous~ ~
expression Knock-out Promoter
analysis
,~ ,~ mice
Recombinant Mutantprotein
protein
<~
Testfunction(s)
purification of enzymes with the desired properties
from microbial or viral sources. Although they are
all important tools, restriction endonucleases are of
particular importance.
Restriction endonucleases can be thought of as
forming part of a bacterial immune system. 3~These
enzymes specifically recognize and cleave DNA
sequences of invading foreign (ie, viral) DNA,
while leaving their host DNA, previously protected
by specific methylases, intact. Most restriction
enzymes recognize palindromes, DNA sequences
with an internal plane of complementarity (eg,
GAATTC or GGATCC) and are named for the
organism from which they are purified (ie, BamHI
for the first restriction enzyme purified from Bacil-
lus amyloliquefaciens H). Literally hundreds of
different restriction enzymes with different site
specificities are now available, in many cases now
isolated from cloned genes introduced into more
common laboratory bacteria, such as Escherichia
coli. With a restriction enzyme and a ligase, an
enzyme capable of joining two pieces of DNA
together in a 5'-3' phosphodiester linkage, one can
make a recombinant DNA molecule by joining
together two or more pieces of DNA, which were
originally not contiguous.
Today, the freezer of any molecular biology
laboratory will contain kinases (which add phos-
phate groups onto the '5'-hydroxyl end of DNA),
polymerases (which copy DNA or RNA from
primed or unprimed templates), phosphatases
216
(which remove terminal phosphate groups), DNA
exonucleases (for controlled digestion from ter-
mini), and ligases. In addition, reverse transcriptase
is particularly important, being an enzyme capable
of generating a complementary DNA (cDNA) copy
of a primed mRNA template.
Vectors
The ability to make recombinant DNA mol-
ecules would have remained an intellectual curios-
ity without the ability to amplify, or make many
copies of the recombinant DNA. This was achieved
by using vectors, autonomously replicating ve-
hicles for the propagation of DNA. The most
commonly used vectors in molecular biology re-
main plasmids. A plasmid is a small, circular,
autonomously replicating DNA. 3~Laboratory plas-
mids contain an origin of replication, a dominant
selectable marker (usually an antibiotic resistance
gene) and a "polylinker" or multiple cloning site.
The latter is a string of unique restriction endonucle-
ase sites, into which can be introduced foreign
DNA, most easily through ligation of cohesive,
compatible ends. Plasmids may be used simply for
the amplification and propagation of recombinant
DNA sequences, to aid in sequencing these se-
quences, or to express the protein-encoding infor-
mation residing in the DNA sequence. For the latter
two applications, appropriate positioning of the
insert DNA with respect to elements contained
within the plasmid vector is required. Plasmids are
usually transferred into host cells for amplification,
propagation and expression; if the transfer is into
bacteria or yeast, the procedure is known as
transformation, whereas transfer into cultured cells
is known as transfection.
Bacteriophages are viruses that infect bacteria,
and can also be used as recombinant DNA vectors.
Insert DNA replaces non-essential sequences in
these linear vectors to make recombinant phage.
The use of phage can bring technical advantages
over the simpler plasmid systems, particularly with
respect to the size of the inserted DNA region.
Phage/plasmid combinations known as cosmids
allow further increases in the size of foreign DNA
that can b e "cloned", or stably propagated in pure
form. 31 The yeast or bacterial artificial chromo-
some (YAC or BAC) offer further increases in the
maximum allowable size insert, up to 300 kbp; 32
WILLIAM R SHEFFIELD
Libraries
An unusual and powerful feature of molecular
biological approaches is the use of libraries or
"clone banks." No one would have expected a
protein chemist to say, "I want to purify coagula-
tion factor VIII. Therefore I will purify all plasma
proteins and then determine which one I want." Yet
this is the essence of screening a library for a gene
sequence. Libraries are vector preparations, which
instead of containing a single kind of insert DNA,
are ideally made up of hundreds of thousands of
independent insert DNA sequences, which taken
overall, represent the total genetic diversity of the
starting material. When transferred to bacteria
under appropriate conditions, each bacterial cell
will take up a single plasmid, containing a single
cDNA insert, or "clone". The challenge in working
with libraries lies in devising effective means of
screening them to identify the potentially rare clone
that encodes the gene of interest. Once clones have
been isolated, or even enriched, they can be readily
biologically amplified by growing up the bacteria
or bacteriophage harbouring the foreign DNA, and
the process is repeated until a pure clone is isolated.
Libraries are one of two general types: cDNA or
genomic. The genetic starting material for the
construction of a cDNA library is mRNA, whereas
that used for a genomic library is genomic DNA,
partially digested with a restriction endonuclease to
cleave it into pieces of manageable size. As edited
versions of genes, cDNA clones are ideal for those
wishing to derive the primary'sequence of a protein
of interest, or to be able to express it in a
heterologous system. Conversely, if the investiga-
tor needs access to the promoter region or intron/
exon boundaries, a genomic clone is essential.
Selection of the cell or tissue from which to
make cDNA is an essential first step in library
construction. Indeed, the first cDNAs to be cloned
were those corresponding to mRNAs that predomi-
nated in certain cell types: globin in reticulocytes,
albumin in hepatocytes, growth hormone from
pituitary cells. After mRNA purification, the total
mRNA population is reverse transcribed, using
reverse transcriptases (RTases) purified from mu-
rine or avian viral sources. This synthesis must be
primed, using a small synthetic oligonucleotide.
Originally, this was done using oligo(dT) homopoly-
mers of 12 to 18 units, which bound to the p01y(A)
tail of mRNAs via A:T base pairing. RTase then
catalyzes the copying of this mRNA template into
MOLECULAR BIOLOGY OVERVIEW
cDNA form. This approach is still used today,
although mixtures of random hexamers can also be
used. At the end of this first strand synthesis, the
resulting RNA:DNA hybrid is converted to double-
stranded DNA by degrading the RNA strand with
RNAse H and replacing it with DNA via the action
of DNA polymerase. After addition of synthetic
oligonucleotide linkers or adaptors specifying re-
striction endonuclease sites, the cDNAs can be
inserted into an appropriate vector. Many variations
on this theme have been developed, including the
enrichment of target mRNA sequences by "subtract-
ing" or removing ubiquitously transcribed mRNAs,
and directional approaches in which the first strand
cDNA is provided a 3' homopolymeric C or G tail
through the action of terminal deoxynucleotide
transferase. The cDNA can now be manipulated in
a directional manner, and inserted into whatever
vector (plasmid or phage) is appropriate. 33
Genomic library construction is simpler in that
no synthesis is required, but more complex in that
larger sized pieces of DNA are manipulated. Total
genomic DNA preparations are made and restricted
with enzymes such that only partial digestion is
achieved. This means that a collection of DNA
fragments of 15 to 30 kilobase pairs might be
generated, with, for example MboII-cohesive ends,
but containing within them some uncut MboII sites.
These DNA fragments; too large to be inserted into
a plasmid, can be used to produce recombinant
bacteriophages through ligation of the left and right
"arms" of the digested linear phage genome. After
incubation with packaging extracts that assemble
these recombinant viral DNAs into infective viri-
ons, the phage are used to infect bacteria, and to
produce a primary lysate, or phage stock, contain-
ing the total library. Although it is tempting to
amplify this library by repeated infection, too many
rounds of this process will make the/library less
representative, in that some recombinant clones
encoding parts of the genome could have growth
disadvantages over others. 34
Hybridization
The ability to form hydrogen-bonded, comple-
mentary duplexes, is central to the molecular
genetic flow of information in the cell. In addition,
this property of DNA and RNA has been exploited
for technical reasons. Unlike most proteins, DNA
duplexes can be thermally or chemically denatured
and renatured relatively easily, and the same holds
217
true for RNA-DNA and RNA-RNA interactions.
This means that a radiolabelled DNA molecule can
be used to find its complementary DNA or RNA
sequence, provided that the way has been cleared
by denaturing, inter- and intra-strand separation of
the target nucleic acid. A simple use of this
principle lies in the use of oligo d(T) primers to
hybridize to the poly(A) tract of mRNA referred to
above.
More complicated interactions involve the use of
single stranded DNA probes, radiolabelled by modi-
fication reactions that typically use 32p-substituted
terminal or internal phosphate groups. Suppose that
one had isolated a clone from a liver cDNA library
corresponding to a new protein, and wanted to
know two things: (1) In which other tissues is the
mRNA transcribed; and (2) How big is its gene?
Both questions can be answered, using either an
RNA (Northern) or DNA (Southern) blot. 35,36In the
case of the former, mRNA from each tissue is
separated, in separate lanes, by formaldehyde/
agarose electrophoresis, and transferred to a nitro-
cellulose or nylon membrane. Following cross-
linking to fix the RNA to the blot, the labelled
cDNA probe is allowed to bind to its target. After
the performance of washes under conditions that
minimize non-specific binding, autoradiography is
used to visualize the presence, and size of the
transcript, detected by virtue of its binding the
probe. DNA blotting is analogous, except that
restriction enzymes are used to cut the high molecu-
lar weight DNA. Although the great diversity of the
genome prevents individual bands from being seen
by staining, by hybridization a discrete pattern of
bands emerges; if a full length cDNA was used, it
will bind to all fragments containing exon se-
quences. Adding the size of all hybridizing frag-
ments together gives a reasonable estimate of the
size of the gene, less upstream control elements.
A specialized application of Southern blotting is
known as DNA fingerprinting. 37 This technique
exploits the presence in the genome of variable
number of tandem repeat (VNTR) sequences. These
are di- and tri-nucleotide sequences of unknown
function found at multiple and diverse sites, and
present at each site in 2 to 20 copies. The high
degree of variability in VNTRs means that the use
of several VNTRs with one or more probes on
Southern blots yields a pattern that is distinctive for
each human, and is invaluable in family and
forensic studies.
218
Library Screening
The same principle used in Northern and South-
ern blotting allows libraries to be screened by
hybridization. Again using our novel liver cDNA as
an example, the same radiolabelled probe can be
used to screen a genomic phage library. At appropri-
ate multiplicities of infection, the phage will form
lytic plaques on lawns of bacteria on agar plates.
Oriented nitrocellulose filters laid on these plates
rapidly bind DNA and protein from the plaques,
and, once fixed, provide a membrane surface
analogous to the Southern blot. After hybridization
and autoradiography, zones containing phage with
the gene insert can be excised from the plate, and
the phage eluted and used to reinfect bacteria, this
time at lower plating densities. After several rounds
of this process, a phage plaque derived from a
single recombinant phage clone can be isolated? ~
What then; can be used to probe a cDNA library?
If portions of the protein sequence can be deter-
mined by protein sequencing, degenerate synthetic
oligonucleotide primers can be synthesized. These
mixtures of oligonucleotides contain all possible
choices for the codons specifying the string, or
strings of amino acids, some of which will bind to
the appropriate cDNA. If two separate stretches of
amino acid sequences are known, it is now possible
to make a specific probe using the polymerase
chain reaction 39 (PCR; discussed below).
If the cDNA library has been cloned into an
expression vector, a specific antibody to the protein
of interest can be used to probe the library. The
reason this is possible is because the vector pro-
vides a promoter and prokaryotic initiation of
translation signals; some also encode a portion of a
bacterial protein, followed by unique restriction
sites. Introduction of the cDNA library into this site
will result in some of the cDNAs being fused in
frame to the bacterial protein, thus generating a
fusion protein capable of being recognized by the
antibody. In the case of the widely used Xgt11
phage expression vector, the bacterial protein is the
enzyme [3-galactosidase, and the [3-galactosidase
fusion protein will be found in the plaques, and
bind to nitrocellulose filters. 4~With the continuous
evolution of cloning technology, it has now become
possible to isolate cDNAs corresponding to pro-
teins for which no DNA or antibody probes exist,
by transforming or transfecting pools of clones into
cells that will express the biological activity of the
WILLIAM P. SHEFFIELD
protein of interest. This approach was used to clone
the thrombin receptor. 41
Differential display of mRNA is another tech-
nique in which no prior knowledge of the transcript
is required for partial cloning. 42 After RNA isola-
tion from cells treated with a given drug or
stimulus, and from control cells, oligo dT primers
with an additional 3'C, G, or T are used separately
to reverse transcribe the preparations. Each cDNA
population is then subjected to PCR using random
primers in the presence of radioactive nucleotides,
and the resulting heterogenous population of prod-
ucts is resolved on a sequencing gel. Display
products that are clearly up- or down-regulated by a
given treatment are then identified, excised from
the gel, PCR-amplified, and sequenced. Compari-
son of the DNA sequence of the partial cDNAs
identified in this manner to known sequences in
DNA databases is then used to determine if a novel,
regulated gene has been discovered.
DNA Sequencing
The ability to clone and manipulate DNA would
have had far less impact had it not been for the
development of methods to determine the base
sequence of any piece of DNA. Two equally
effective systems, chemical (Maxam-Gilbert) 43 and
enzymatic (Sanger) were developed; today the
latter system is by far the most commonly used. 44
In it, single stranded DNA or double-stranded
plasmid DNA made effectively single-stranded by
chemical denaturation serves as the template. A
small synthetic oligonucleotide primer of 17 to 20
nucleotides is annealed to a site in the vector just 5'
to the cDNA, and polymerized by a DNA polymer-
ase in the presence of radiolabelled oi" fuorescent
nucleotide triphosphates. A phage T 7 DNA polymer-
ase chemically modified to enhance its polymerase
activity and minimize its exonuclease activity is
widely used for this purpose. The other key ingredi-
ent o f t h e DNA sequencing reactions are dideoxy-
nucleotides that serve as chain terminators because
they lack a 3' hydroxyl to which the next nucleo-
tide can be attached in polymerization. The an-
nealed primer-template preparation-is split into four
separate reactions for polymerization, with each
sub-reaction containing one of the four dideoxy
chain terminators. For instance, the A reaction
contains dCTR dGTP, dTTP, arid a mixture of
deoxy- and dideoxy-ATE The ratio of the latter is
adjusted such that some of the elongated chains
MOLECULAR BIOLOGY OVERVIEW
stop at any given A. Electrophoresis of these
populations of radioactive single-stranded DNA
molecules, using high resolution urea-acrylamide
electrophoresis capable of resolving DNA mol-
ecules differing by a single base, is then used.
Electrophoresis of the four reactions side-by-side
produces a sequencing ladder that can be read by
visual inspection of autoradiograms, or, as is increas-
ingly the case, by computerized recovery of the
information from an automated system that uses
fluorescently tagged nucleotides. Various strategies
have been devised to "read-through" areas of DNA
with strong intrachain associations that would
otherwise stop or impair the processive movement
of the sequencing polymerase.
Heterologous Expression Systems
Isolation of a cDNA furnishes the investigator
with all the nucleic acid information required to
produce the protein in an expression system, pro-
vided that the information is presented in an
appropriate manner. The systems are said to be
heterologous because they involve the expression
of foreign DNA, and because they generally result
in expression of a protein in a milieu in which it
would otherwise not be found. This latter feature
eliminates the need to separate the recombinant
protein from the endogenous version that would
otherwise be present in a homologous system.
The use of Xgt-ll expression cloning systems
results in bacterial expression of a [3-galactosidase
fusion protein, which provides at a minimum a
source of antigen of the cloned protein of interest.
In some cases, the fusion protein acquires the
biological activity of t h e cloned fusion partner.
This fusion approach has been incorporated into
numerous plasmid expression vectors that are easier
to use than a phage system. The advantage of this
approach is that native or hybrid bacterial promot-
ers and sites of initiation are used, and the presence
of the bacterial portion of the protein can stabilize
the fusion protein against degradation. Unfortu-
nately, whether fused or unfused expression of
foreign cDNAs in bacteria is used, the resulting
product is often insoluble and nonfunctional. Al-
though in some cases this "aggregation body" can
be isolated, and the protein solubilized and re-
folded, the process can be difficult and empirical.
Irrespective of this potential outcome, bacteria are
incapable of performing post-translational modifi-
cations such as N-linked glycosylation and ",/-car-
219
boxylation, and generally cannot be relied on to
promote native disulphide bond formation in an
efficient manner. These problems can sometimes
outweigh the ease and low expense of expression in
bacteria. 45
Yeast expression systems generally fare better at
folding foreign (especially mammalian) proteins,
and are capable of many post-translational modifi-
cations, including N-linked glycosylation. How-
ever, the commonly used baker's yeast Saccharomy-
ces cerevisiae can hyperglycosylate, adding up to
80 sugar subunits to sites that generally have only
10 to 15 in their natural setting; qualitative differ-
ences in glycosylation are also found. New yeast
systems, such as the methylotropic Pichia pastoris
may overcome this problem. 46 Moving further up
the evolutionary scale, expression of heterologous
proteins in insect cells, using a system derived from
baculovirus, can provide high yields of well-
folded, post-translationally modified products. The
technical difficulty Of working with this system has
recently been reduced. 47
Mammalian cell expression systems are undoubt-
edly the closest experimental system to the native
site of production of human proteins. 48 In contrast
to bacterial systems, if a full-length cDNA is
available, it can be inserted into a mammalian cell
expression vector without extensive manipulation.
These vectors provide appropriate strong enhancer/
promoter sequences and polyadenylation signals on
opposite sites of a cloning site, and can provide
non-translated intron sequences that add to the
efficiency of expression. Transfection of these
expression vectors in cell culture, using chemical
or liposome-mediated uptake results in transient
expression of recombinant products, for instance in
the widely used SV40 virus-transformed simian
kidney COS cell line. The ~tg levels of protein
produced in this rapid expression system are often
sufficient for some analyses. Obtaining more pro-
tein necessitates the generation of permanently
transformed cell lines. In this case, relatively rare
cells in which the plasmid expression vector has
inserted itself into the genome must be selected,
usually through cotransfection or provision on the
sameexpression vector of genes conferring resis-
tance to antibiotics (typically G418 or hygromy-
cin). Although generation of stable cell lines is
expensive and time consuming, it is usually pos-
sible to select high expressors, cell lines in which
the expression vector has fortuitously integrated in
220
a genomic location conducive to high expression.
Continuous propagation of such lines can yield
milligram levels of recombinant protein, and is the
technique used on an industrial scale to produce
recombinant factor VIII for treatment of hemo-
philia A. 49
Mutagenesis
If an acceptable system has been found for
heterologous expression of the protein product of a
cDNA, then molecular techniques can be used to
alter single or multiple codons of that cDNA.
Following confirmation that the desired changes
have been made by DNA sequencing, the mutated
cDNA can then be expressed, and the altered
protein assessed for any resulting changes in biologi-
cal function. This site-directed mutagenesis 5~ is
generally achieved through the use of a synthetic
DNA oligonucleotide that is complementary to a
portion of the cDNA in a plasmid vector, with the
exception of one or more bases flanked on either
side by 9 to 15 unchanged bases. After denaturation
of the plasmid, these regions of complementarity
are usually sufficient to permit binding of the
mutagenic oligonucleotide to its target site in spite
of the internal mismatch. This oligonucleotide is
used to prime synthesis of the rest of the other
strand of the plasmid. When the plasmid with the
internal mismatch is used to transform bacteria,
progeny formed after replication contain either
mutant or parental sequences. A variety of strate-
gies exist for increasing the proportion of mutant
plasmids over parental, such that DNA sequencing
of a handful of inserts of the resulting plasmids
yields the desired mutant. Other ways to alter
cDNAs include truncation using processive exo-
nucleases to perform controlled digestions, for
instance on the 3' end of the cDNA to make a
family of proteins with the same N-terminal, but
progressively shorter C-terminal regions, or linker-
scanning, in which a dipeptide is introduced at
different points Within the cDNA. In all cases, the
resulting mutant proteins are assessed for loss of
function that ties the mutation to an important site
on the protein. Purification of recombinant proteins
in any system has been simplified by the develop-
ment of affinity tags, short stretches of amino acids
which, when incorporated into the recombinant
protein, confer the ability to bind to affinity or
immunoaffnity columns.
Of course, natural mutations are also found,
WILLIAM P. SHEFFIELD
including those that give rise to human genetic
disease. The techniques of molecular biology have
been applied to characterize the genetic lesion in
numerous genes and kindreds. Of particular use in
this regard is the technique of PCR. Briefly, this
amplification technology makes use of two oppos-
ing gene specific primers, a heat stable polymerase
(from Thermus aquaticus [Taq] or other extremo-
philic microorganisms), and an automated thermo-
cycler. By alternating between temperatures, this
process uses the polymerized DNA product of one
cycle as a template for the production of more DNA
product in the next, and achieves a geometric
amplification. The extraordinary power of this
technique is explained in detail in a recent review in
this journal. 51
Transgenic Mice
Transgenic mice are animals whose genome has
been stably altered by genetic manipulation. In a
sense, the use of transgenic mice can be seen as
either heterologous expression or mutagenesis on a
grand scale. 52 Two routes of transgenesis are used:
microinjection of fertilized murine ova, and genera-
tion of chimeric animals following genetic manipu-
lation of pluripotent embryonic stem cells. The
former, which is technically simpler and was
developed first, uses linearized plasmid DNA con-
structs in which a cDNA has been positioned
downstream of an enhancer/promoter. After micro-
injection of this material, some ova will integrate
multiple copies of the constrfict as a tandem array.
Transfer of the ova to foster mothers leads to the
birth of offspring, some of whom will have inte-
grated the DNA into all of their cells. Some of these
animals will also express high levels of the encoded
product; mice transgenic for human growth hor-
mone, to cite an early example, are double the size
of non-transgenic littermates. This form of transgen-
esis is ideal for adding something to the mouse
genome, for instance, high level oncogene expres-
sion to develop an in vivo carcinogenesis model.
Another example would be the secretion of clotting
factors into the milk of transgenic mice. Transfer of
this technology to larger dairy animals could one
day provide an economical, non-blood product
source of factor concentrates) 3
Taking something away from the mouse genome
requires additional manipulation. First, genomic
clones of the target gene are modified such that an
essential element is replaced by a minigene encod-
MOLECULAR BIOLOGY OVERVIEW
ing antibiotic resistance. Next, embryonic stem
cells are transfected with these constructs, and
stable integrants are selected. A subset of these
clones undergo homologous recombination to re-
place the endogenous copy of the target gene with
the engineered, disabled one. Embryonic stem cell
lines harbouring this targeted change are grown up
and injected into the blastocyst of developing mice
embryos. Resulting offspring are chimeric in that
some of their cell lineages derive from the engi-
neered cells. Breeding these animals produces
offspring in which one or both copies of the gene of
interest have been disabled. 54 Examples of these
engineered mutant mice now abound; a recent
example involves the "knockout" of the procoagu-
lant tissue factor geneY
Promoter Studies
The intense scientific interest in gene regulation,
particularly at the transcriptional level, has spawned
a variety of molecular biological techniques for
studying promoter activity. The initial location of
promoter/enhancer sequences is often achieved by
an approach that is the reverse of what is used to
produce heterologous expression of cDNAs. In-
stead of inserting the coding region into a vector
providing the promoter, reporter gene expression
vectors require the insertion of putative promoter
sequences upstream of a readily detected cDNA
such as that of chloramphenicol acetyl transferase,
luciferase, or naturally fluorescent proteins. A rea-
sonable approach would be to position as many
kilobase (kb) of upstream genomic DNA that is
available in such a construct, then use deletional
rnutagenesis to whittle this down to a minimum
functional promoter. The expression plasmids are
transfected into cell lines, and cell supernatants or
lysates are analyzed for the presence of the indica-
tor protein or its activity. A more ambitious varia-
tion on this theme, aimed at determining the tissue
specific nature of promoter sequences in vivo, is to
generate transgenic mice using the reporter expres-
sion plasmid, and then examine various tissues and
cell types for the presence of the reporter protein.
Conceptually similar studies can be performed in
vitro, using nuclear extracts) 6 These either involve
the generation of transcripts in an in vitro transcrip-
tion system, in which care must be taken to control
for nonspecific transcription arising from the disrup-
tion of the normal context of template DNA, or rely
on electrophoretic mobility shift analysis (EMSA). 57
221
In the latter technique, a radiolabelled putative
promoter sequence (eg, one excised from a ge-
nomic clone) is incubated with a nuclear extract. If
transcription factors bind to the labelled DNA to
form a complex, this complex will display dimin-
ished migration upon subsequent non-denaturing
electrophoresis. This shift in mobility can be fur-
ther dissected by competition with specific oligo-
nucleotides corresponding to known transactivator
binding sites, or by producing a "super-shift" when
specific antibodies to known transcription factors
are added to the reaction.
Determination of transcription factor binding
sites can also be aided through the use of deoxyribo-
nuclease I (DNase I). DNAse I digestion of mini-
mally perturbed genomic DNA, followed by South-
ern blotting to determine the site of DNase I
hypersensitivity, is useful in mapping sites of
nucleosome clearing and trans-acting factor bind-
ing.. This approach has been useful in finding
important regulatory sites that are unusually distant
from the genes they control, for example, the Locus
Control, Region (LCR), which lies 5-25 kb up-
stream Of the first globin gene whose activity it
regulates in the globin gene cluster. 58 DNase I will
also cleave radiolabelled DNA constructs in vitro,
on average every few base pairs to generate a
ladder. Introdncfion of increasing amounts of nuclear
extracts into these reactions will alter the pattern, as
stretches of DNA that would otherwise be cleaved
by DNase become protected by DNA-binding
proteins. Conceptually similar schemes use other
DNA modifying agents such as methylating agents,
and are also useful for this kind of fine mapping of
modification-sensitive transcription factor binding.
CONCLUSION
This article is first and foremost an overview, in
that it is impossible to cover in a few pages an area
of biology that is the subject of large textbooks and
technical manuals. Nevertheless , the concepts and
examples of techniques given above shotild pro-
vide the reader with the broad outlines necessary to
understand other molecular techniques and issues
that have not been addressed here. For instance, the
Human Genome Project,59 which aims to sequence
every nucleotide in the human genome by 2005, is
analogous to the smaller scale DNA sequencing
and DNA fragment ordering discussed here. The
yeast two-hybrid system6~becomes understandable
222
as a means of detecting protein-protein interactions
by transcriptional activation. Phage display sys-
tems can now be thought of as "classical" library
screening in reverse, where instead of probing
immobilized clones with a free probe, the library is
allowed to flow past an immobilizedprobe. 57
Without a doubt, the years to come will see a
continued integration of molecular biological infor-
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