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chapter 18
Molecular biology
Steve Hood

18.1 Introduction
Molecular biology is now a recognised facet of modern pharmaceutical research,
from the discovery of novel compounds to the pre-clinical evaluation of new drugs.
The basic principles and common protocols are now incorporated in many under-
graduate teaching programmes and the introduction of pure molecular biology
degree courses has established a skill base within industry and academia.
To fully cover ‘what you should know’ could run into several books. This chapter
aims to give an overview of the underlying science of molecular biology and then
highlights its uses within the field of drug metabolism.
This chapter has been divided into three sections. The first section covers the
underlying theory that is the basis of the molecular biology used in a modern drug
metabolism lab. The remaining sections address the two major questions that
molecular biology can help to answer: which enzyme metabolises my drug and
does my drug induce or suppress the metabolism of another coadministered drug.

18.2 Basic molecular biology

In 1944, Oswald Avery provided evidence that DNA may play a role in the genetic
information of the cell, but it was not until 1953 that Watson and Crick (with
Franklin and Williams) proposed their model for the structure of DNA, and the

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326 Steve Hood

science of molecular biology was born. It was realised that this simple, self-
replicating molecule comprising four bases and a sugar–phosphate backbone could
encode for all the diverse proteins found within a living organism. Because the
structure consisted of two antiparallel strands of nucleotides, such molecules could
be unwound and duplicated, thus explaining the principles of heredity and cell
division.
The backbone of the nucleic acid is made up of a chain of ribose sugar units
linked by phosphate residues in the 3- and 5-positions of the ring. In RNA the sugar
exists as a ribose residue while in DNA the sugar is reduced further to the
deoxyribose form. Attached to the 1-position of the ribose is one of the four bases,
adenine, guanine, cytosine and thymine (or uracil in RNA). A cytosine residue can
pair with guanine residue on the other strand by means of three non-covalent
hydrogen bonds while adenine and thymine pair with two such bonds. It therefore
requires more energy (as heat) to break a GC bond as is required for an AT bond,
and this is an important factor in techniques which require strands to hybridise to
each other. This is shown in Figure 18.1.
The genetic information is arranged into genes, each gene coding for a single
protein. Eukaryotic genes have some common features in their structure. At the front
(50 -end) of the gene there is a region of DNA called the promoter. This is the on/off
switch for the gene and can be regulated by a series of proteins inside the nucleus of the

5′ 3′

T A

C G sugar

A T G

base

C G

DNA helix DNA uncoiled

Figure 18.1 The base pairing of the DNA double helix.

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Molecular biology 327

cell. The genetic information of the gene is grouped in regions called exons. These
exons are separated by regions of non-coding ‘junk’ DNA (introns) which are not
involved in the production of the final protein. In prokaryotic genomes, where space is
limited, there are no introns (see Figure 18.2). When the gene is activated, RNA
polymerase binds to the promoter and proceeds to make a single-stranded RNA copy
of the coding strand of the DNA. This process is known as transcription. The introns
are then cut out of this new RNA strand in a process called splicing. The resulting
strand contains information derived from the exons only and is called messenger RNA
(mRNA). The mRNA is then exported from the nucleus where it is used as a
blueprint to make the protein. This process is known as translation and is performed
by the ribosomes. The bases on the mRNA are read in groups of three, known as
codons, each group coding for an amino acid or a ribosomal command (e.g. ‘start’ or
‘stop’ translation). The ribosomes run along the mRNA molecule with the growing
protein chain until the stop signal is reached, when the ribosome drops off the chain
and the protein is finished. The single-stranded RNA molecule is prone to rapid
degradation and therefore the amount of protein produced is closely regulated at the
transcriptional level. The processes of transcription and translation are collectively
known as gene expression.
The workhorse of the molecular biologist is the bacterium and as this is a
prokaryotic organism, it cannot perform intron splicing. Therefore genomic DNA
cannot be used for expression in these cells as the introns would also be translated
leading to the production of a ‘nonsense’ protein. The instability of RNA limits its
use as a template for expression in bacteria and so another form was required.
Virologists, working on a group of viruses that carried their genetic information as
RNA (the Retrovirus), discovered that the life cycle includes a conversion of the single-
stranded RNA into a double-stranded analogue. The enzyme responsible for this

Promoter Intron Exon 3′UTR

DNA

Transcription

mRNA AAAAAAA

Translation Reverse transcription

TTTTTTT
AAAAAAA

cDNA
PROTEIN

Figure 18.2 The flow of genetic information from DNA (and cDNA) to protein.

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328 Steve Hood

step was isolated and named reverse transcriptase. By using this enzyme, it is possible
to convert eukaryotic mRNA into a more stable double-stranded molecule with the
properties of DNA. This molecule is known as complementary DNA (cDNA) and is
equivalent to the corresponding genomic DNA but without the introns or pro-
moter. This can then be used for expression in a prokaryotic (or eukaryotic system).
The relationship between DNA, RNA, cDNA and protein is shown in Figure 18.2.
The underlying principle in genetic manipulation is that if you insert a fragment of
DNA into a host cell, it will be duplicated as the cell divides and therefore you will
increase the number of copies of your fragment. In order to perform this amplifica-
tion, the DNA must be inserted into a suitable part of the host cell’s genetic
material so that it will be duplicated along with the endogenous DNA during cell
division. DNA suitable for the insertion of foreign material is known as a vector;
the most commonly used vectors are plasmids.
Plasmids are small (2–3 kb) loops of DNA found in bacteria and yeast. They were
first discovered when it was noticed that bacteria could pass antibiotic resistance
from one colony to another. This was shown to be mediated by plasmids containing
genes for enzymes that de-activated the antibiotics. While plasmids were being
investigated, other groups had isolated enzymes that cut DNA at specific sequences
(restriction endonucleases) and other enzymes that could rejoin these cuts again
(DNA ligases).
The principles of cloning are shown in Figure 18.3. Both vector and insert are cut
with the same restriction enzyme, purified and then incubated together with a
ligase that reforms the backbone of the plasmid. The resulting recombinant DNA is
inserted into bacterial cells which then multiply. Each cell can contain 50–100

R
R
R + R Recombinant DNA
R
Vector Insert +

Bacterium
Vector
multiplication
Transformed Each colony derived from
Cell single recombinant
bacterium
division molecule

Plate out on
selective media

Figure 18.3 Cloning and selection of recombinant DNA.

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Molecular biology 329

copies of the recombinant plasmid and can duplicate every 20–30 minutes. Most
plasmids have been engineered to contain one or more antibiotic resistance gene
which protects the cells when they are grown on selective agar plates. Once selected,
the cells can be grown up in liquid culture and the plasmids re-isolated, purified
and studied further.

18.3 Which enzyme?

The identification of the route of metabolism is a key part of the regulatory package
of a new drug compound and this work can be approached from two directions.
The first, and simplest option, is to use heterologous systems containing a single-
expressed enzyme and examine if the drug is turned over in such a system. This
approach allows the kinetics of the drug to be examined in isolation from any
competing reactions.
The alternative approach is to look at the complete metabolism system, e.g.
human liver microsomes, and to knockout single enzymes. By determining the
metabolism that does not occur in the inhibited state, it is possible to extrapolate
the role of that isoform in the metabolism of the compound. This approach can be
achieved by the use of specific chemical inhibitors (e.g. ketoconazole for 3A4) or
inhibitory antibodies raised to specific epitopes on the target enzyme and is
discussed in other chapters in this volume. However, molecular biology will allow
us to generate transgenically altered animals where the enzyme of interest has been
removed (currently only available in mice).

18.4 Expressed enzymes

1 8 . 4 . 1 I N TR OD U C TI O N

Heterologously expressed enzymes have become important tools for the study of
Phase I and Phase II drug metabolism. While the majority of the work has been
focused on the cytochrome P450 family, the approach has successfully been utilised
for the expression of sulphotransferases (STs), flavin mono-oxygenases (FMOs) and
UDP glucuronosyltransferases (UGTs). The role of these enzymes in the activation
and detoxification and clearance of xenobiotics make them important targets for
understanding and predicting drug metabolism. In order to characterise the activity
of these enzymes, it is necessary to produce quantities of pure protein, both for
catalytic activity studies, the production of metabolites (bioreactors) and for the
production of antibodies which can be used to characterise the levels of expression of
the protein in other tissues.

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330 Steve Hood

Traditionally this has been achieved by isolating and purifying the enzymes
from hepatic microsomes prepared from animals that had been pre-treated with
inducing agents. The purification procedure involves a series of fractionation
steps involving a range of gel-filtration and affinity columns. However, even
after extensive purification, it is still not possible to separate closely related but
distinct isoforms. This approach is not suitable for the isolation of human
isoforms as the availability of human tissue for the preparation of microsomes
is limited, and the induction of donors to enrich the levels of poorly expressed
isoforms is both unethical and impractical. This problem has been addressed by
several groups, and human drug-metabolising enzymes have been expressed in
bacteria, yeast and cultured cell lines. This is known as heterologous expression
as the gene from one species (e.g. man) is expressed in another (e.g. Escherichia
coli).
Heterologous expression is extensively described in Methods in Enzymology (1996)
vol. 27.

1 8 . 4 . 2 W HY U S E E X P R E S S E D E N Z Y M E S ?

Expressed enzymes are becoming increasingly popular as tools for metabolism

studies and are used to replace human liver microsomes (HLMs). Their utility can
be identified by the following properties:

User-friendly – The enzymes are supplied as membrane preparations or purified

protein and can therefore be used directly in existing metabolism incubation
protocols.
High activity – Because each enzyme is overexpressed in the heterologous system
there is a high specific activity. This is significant for isoforms expressed at a low
level in human tissue. Expressed enzymes do not display inter-individual variation
seen with HLMs.
Single enzyme in each batch – The lack of any competition between different isoforms
within the heterologous system allows a more accurate assessment of the contribu-
tion of that isoform to the metabolism of the test compound.

The disadvantages of the systems should also be noted:

Environment – Heterologously expressed protein is not in the native environment

(especially in the cell membranes of E. coli) and therefore may exhibit abnormal
catalytic activity.
Specificity – Over-expression of enzymes can lead to a loss of substrate specificity and
thus gives misleading profiles.

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Molecular biology 331

1 8 . 4 . 3 C H O I C E O F EX P R E S S I O N S Y S T E M S
The expression systems commonly used within industrial research can be divided
into four main groups, determined by the host cell type. These are bacterial-based,
yeast-based, insect cell-based and mammalian cell-based systems. The selection of
the optimum cell system for the expression of drug-metabolising enzymes depends
on a variety of factors. Some considerations for the expression of P450s are listed
below and summarised in Table 18.1.
Mammalian P450s are membrane bound to either the SER or the mitochondrial
inner membrane and require phospholipids to function normally. The NH2 -
terminus of the protein contains a signal recognition/insertion sequence which
both targets and anchors the protein to the appropriate membrane. This sequence
does not play any further role in the catalytic activity of the enzyme, as the enzyme
is still functional if the region has been deleted.
The cytochrome P450 enzyme requires a source of reducing agents and in the
SER this is provided by NADPH-cytochrome P450 reductase (and in some cases
cytochrome b5 ). In the mitochondria, this is replaced by a two-component system
consisting of adrenodoxin and NADPH-adrenodoxin reductase. A purified enzyme
can be reconstituted to its active form in vitro by the addition of NADPH-
cytochrome P450 reductase and phospholipids. The species from which the reduc-
tase is isolated is not crucial and heterologously expressed recombinant reductase
has been successfully used in various reconstitution experiments.
Cytochrome P450 contains a non-covalently bound haem molecule and the
expression system must be able to produce and insert the haem into the nascent
enzyme if functional expression is to be achieved. There is therefore a requirement
to supplement the culture with a suitable haem source such as D-amino levulinic
acid or hemin Cl.

Table 18.1 A comparison of expression systems and their suitability for P450 expression

Expression system

Properties Mammalian Insect Yeast Bacteria

Vector system Vaccinia Baculovirus Plasmid Plasmid

Ease of culture þþ þþ þþþ þþþ
Expression levels (Hlms ¼ 100%) 25% 200% 75% 300%
Haem incorporation þþ þþ þþ þþþ
Microsomal membranes 3 3 3 5
Endogenous P450 P450 P450 Puteroredo
reductase reductase reductase reductase xin
Endogenous P450s þþþ þþ þþ þ

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332 Steve Hood

Cytochrome P450 is a globular protein and the host cell must have the ability to
fold the protein correctly if a functional enzyme is to be produced.

Expression in bacteria
Bacterial expression
Bacteria, especially E. coli, are a good system for the expression of foreign proteins
as, like yeast, it is well understood genetically and can be manipulated to suit the
specific requirements. The cells can also be grown to high densities with ease.
However, the bacteria have significant structural differences from the eukaryotic
cells. There is no SER or NADPH-P450 reductase although a bacterial version is
present and has been shown to have some activity with heterologous P450s.
Surprisingly, work with E. coli has shown that these differences are not insur-
mountable and several groups have reported the expression of functional P450s in
such a system (see Table 18.2).
Cell types
E. coli
The work horses of bacterial expression are the variants of E. coli that have been
developed for molecular biology. The principal strains used JM109 and DH5a
which are both commercially available from most and can be used for general
cloning work and expression. Another E. coli-derived cell line is BL21(DE3) which
is specifically designed for use with the T7 promotor system. The majority of
bacterial expression listed in Table 18.2 utilises these strains.
Salmonella typhimurium
This is the strain used in the Ames assay for mutagenic compounds and their
metabolites. Traditionally compounds are incubated with rat S9 fraction and the
resulting metabolites tested for their abilities to revert S. typhimurium mutants back
to wild type. By transforming these mutants with a recombinant P450 it is possible
to re-create the assay without the need for S9. This approach has been used to
express human CYP1A2 in T1538.
Bacterial expression protocols
Protocols for the expression of P450s in bacteria are reviewed by Barnes (1996) and
are based on the methods devised by Gillam et al. (1993).

Expression in yeast
Yeast expression
Yeast systems have the advantage of well-characterised genetics and ease of growth
in large volumes. There are also endogenous reductase systems and SER membranes

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Molecular biology 333

with which the expressed proteins can associate. This system has been used success-
fully to produce a wide range of P450s (see Methods in Enzymology, 206, pp. 132–133)
including human CYP2C9, 2C10 and 3A4. The catalytic rates of the expressed
enzymes for specific probe substrates are comparable to those in human liver
microsomes (Masimirembwa et al., 1999).
Cell types
Saccharomyces cerevisiae
This is the most common strain of yeast used for the expression of P450s and other
enzymes. There have been some reports of poor haem incorporation but these are in
a minority.
It is possible to make stable yeast clones where multiple copies of the human
cDNAs are incorporated into the yeast genome. Co-expression of OR and cyto-
chrome b5 is also stably incorporated to give functional expression systems.
Pichia pastoralis
A new system utilising P. pastoralis has recently been developed by invitrogen and
claims to give higher yields than existing systems although this has yet to be tried
for P450 expression.
Yeast expression protocols
Yeast expression protocols are described by Masimirembwa et al. (1999). The
heterologous P450s are inserted into the yeast ER and therefore standard micro-
somal preparation techniques may be employed for their recovery once the cell wall
is broken. This can be achieved by the addition of a yeast lytic enzyme (e.g.
zymolase).

Expression in baculovirus
Insect cell expression systems
The baculovirus expression system is a highly effective system for the large-scale
production of recombinant proteins in insect cells. The major attraction of baculo-
virus as an expression vector system was originally the virus-encoded polyhedrin
and p10 genes.
These genes produce large amounts of polyhedrin and p10 proteins in virus-
infected insect cells in the later stages of the virus replication cycle. The polyhedrin
protein, controlled by the polh promoter, is required in the normal infection cycle to
package virus particles within occlusion bodies or polyhedra, which protect the
virus particles in the environment between susceptible hosts.
The function of the p10 protein is not clear as yet but it is thought to be involved
in polyhedra formation and is controlled by the p10 promoter. Polyhedrin and p10
are not required to maintain an infection in cultured cells in vitro. The polyhedrin

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334 Steve Hood
Table 18.2 Expression of human drug metabolising enzymes in different host cells

Mammalian
COS cells, CHO,
V79, HepG2/
Enzyme Bacteria Yeast Baculovirus Vaccinia b-Lymphoblastoid

CYP1A1 Guo et al., 1994 Eugster et al., 1990 Buters et al., 1995 McManus et al., 1990 Crespi et al., 1993
CYP1A2 Fisher et al., 1992 Eugster and Sengstag, Christopherson et al., Aoyama et al., 1989a; Crespi et al., 1990a
1993 1995 McManus et al., 1990
CYP1B1 Shimada et al., 1998 Fujita et al., 2001 Crespi and Miller, 1999 Crespi et al., 1997
CYP2A6 Kushida et al., 2000 Lessard et al., 1997 Grogan et al., 1995 Yamano et al., 1989a; Crespi et al., 1991
Miles et al., 1990
CYP2B6 Gervot et al., 1999 Gervot et al., 1999 Roy et al., 1999 Yamano et al., 1989b Chang et al., 1993
CYP2C8 Richardson et al., 1995 Srivastava et al., 1991 Zeldin et al., 1995 Veronese et al., 1993; Crespi et al., 1993
Rettie et al., 1994
CYP2C9 Sandhu et al., 1993 Yasumori et al., 1989 Grogan et al., 1995 Veronese et al., 1993; Crespi et al., 1993
Rettie et al., 1994
CYP2C18 Richardson et al., 1995 Goldstein et al., 1994 Crespi and Miller, 1999 Furuya et al., 1991
CYP2C19 Richardson et al., 1995 Goldstein et al., 1994 Christopherson et al., 1995 Crespi and Miller,
1999
CYP2D6 Gillam et al., 1995a Ellis et al., 1992 Christopherson et al., 1995 Tyndale et al., 1991 Penman et al., 1993

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CYP2E1 Gillam et al., 1994 Patten and Koch, 1995 Patten et al., 1992; Crespi et al., 1990b
Tassaneeyakul
et al., 1993
CYP3A4 Gillam et al., 1993 Renaud et al., 1990 Lee et al., 1995 Aoyama et al., 1989b; Crespi et al., 1991
Ball et al., 1992
CYP3A5 Gillam et al., 1995b Fujita et al., 2001 Crespi and Miller, 1999 Aoyama et al., 1989b;
Ball et al., 1992
CYP4A11 Palmer et al., 1993 Imaoka et al., 1993 Imaoka et al., 1993 Crespi and Miller,
1999
UDPGT Ouzzine et al., 1994 Sheen et al., 1998 Ouzzine et al., 1999;
Forsman et al., 2000
SULT Guengerich et al., 1996 Falany et al., 2000
FMO Cashman et al., 1997 Itoh et al., 1993 Haining et al., 1997
MAO Lu et al., 1996 Miller and Rebrin et al., 2001
Edmondson, 1999
NAT Delomenie et al., 1997 Blum et al., 1990;
Yanagawa et al., 1994

Molecular biology
GST Thier et al., 1995 Cho et al., 2001

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336 Steve Hood

and p10 genes can be replaced with foreign gene sequences and thus express
recombinant protein from the polyhedrin (polh) and p10 (p10) promoters.
The insect cell-baculovirus expression system provides a eukaryotic environment
that is generally conducive to the proper folding, disulphide bond formation,
oligomerisation and/or other post-translation modifications required for the bio-
logical activity of some eukaryotic proteins. These modifications are performed
using insect cell-derived sugars and are therefore near-authentic versions of the
original proteins.
The baculovirus insect cell system has been used for the expression of several
P450 enzymes (see Table 18.2) and is the method of choice for the generation of
high-activity microsomes (Crespi and Miller, 1999).
Insect host cell types
Insect cell lines can be obtained from commercial suppliers or from the American
type culture collection (Rockville, MD).
Spodoptera frugiperda
S. frugiperda is the fall army worm and the most common insect cell donor and is
found as the SF9 and SF21 cell lines. These cells are used to grow, maintain and
titre baculovirus stocks and can be used for enzyme expression.
Tricoplusia ni
T. ni is the cabbage looper worm and its cells are smaller and more adaptable to
fermentation as they can be grown to higher density than SF9 cells (Lee et al., 1995).
Drosophilla melanogaster
Drosophilla has been the basis of genetic studies for decades and is therefore well
understood. This has lead to the development of stably transfected (transgenic)
Drosophilla cells. The advantage of these cells is that they do not need a virus system
for expression and are therefore easier to maintain. This has been proposed as a
possible in vivo model for various isoforms but it would be more difficult to extract
pure enzymes due to the multicellular nature and size of the insect (Jowett et al.,
1991).
Baculovirus expression protocols
The handling of insect cells requires cell culture facilities and good aseptic tech-
nique, and the cultures are prone to fungal and bacterial infections. The heterologous
P450s are inserted into the ER membranes of the insect cells and therefore standard
microsomal preparation techniques may be employed for their recovery. The insect
cells are robust and can be cultured in large volume sparged fermenters designed for
bacterial production. This has allowed cultures up to 50 L to be fermented,
harvested and 2 g of microsomal protein isolated from a single batch.
Protocols for generation and maintenance of baculovirus stocks, and the expres-
sion and scale up of insect cell can be found in Hood et al. (1998).

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Molecular biology 337

Expression in mammalian cells

Mammalian expression systems
Mammalian cell systems most closely resemble the in vivo environment of the
enzyme and as such are potentially the most likely to produce the protein in its
functional state.
The main disadvantage is the cells have endogenous P450s which may interfere
with the purification of the enzyme and give a background activity in microsomal
catalytic studies. The cells may also show low expression of the P450s and poor
haem incorporation. Therefore, scale up for isolation of protein can be both costly
and difficult to engineer (Crespi and Miller, 1999).
Cell types and vectors
COS cells
These cells are derived from an African green monkey kidney cell line (CV-1) that
has been immortalised with SV40 viral DNA. This cell line produces the SV40 T
antigen which allows plasmids containing the SV40 origin to replicate and express
in this cell line. The most common vector used is the pCMV system which contains
the promotor from the cytomegalovirus (Clark and Waterman, 1991).
HepG2
These cells are derived from a human hepatoma and are well characterised in the
literature. They can be transformed using a plasmid system, but the main utilisa-
tion has been for vaccinia virus-mediated expression.
These cells are lysed during the expression process and can therefore only be used
for membrane preparations. There is also a risk inherent in the infective nature of
the virus that requires special containment to prevent operator exposure.
V79/CHO
These cells are derived from lung fibroblasts and ovaries of the Chinese hamster,
respectively. The expression is driven by an SV40-containing plasmid which can
result in a stable expression of the enzyme of choice. The non-lytic nature of this
system allows the use of whole cells for toxicology and bioreactor experiments.
NIH 3T3
This is another fibroblast-derived cell line that can be transformed using the
vaccinia virus system.

-Lymphoblastoid
The AHH-1 human
-lymphoblastoid cell line has been transformed using epstein
barr virus (EBV)-based vectors. This system has been used to express single and
multiple P450s and was the basis of the first commercially available recombinant
microsomes (Crespi et al., 1993).

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338 Steve Hood

Caco-2/MDCK
Epithelial cell monolayers are now routinely being used as models of intestinal
permeability for drug candidates, and common cell lines are MDCK, Caco-2 and
LLC-PK. In native intestinal cells, high levels of CYP3A4 are expressed and act as
a metabolic barrier, but the cell models lack such an expression level. Using the
vector systems developed for CYP3A4 expression
-lymphoblastoid cells, Crespi
et al. (2000) have developed cell lines that are metabolically closer to the endogen-
ous gut epithelia.

Expression protocols
The culture conditions vary with the vector:cell line combinations, and protocols
can be obtained from the references cited above. Cells can be used as intact
bioreactors or to be processed to microsomal membranes for use in metabolism
studies.
Care must be taken to minimise the risk of contamination, both from the
environment into the cell culture and, in the cases of the virally transformed cells,
from the cell culture to the operator.

Applications of expressed enzymes

Drug metabolism and enzyme inhibition studies
The principal uses of recombinant enzymes are the determination of route of
metabolism and the assessment of the inhibitory potential of candidate drugs. Both
areas are reviewed extensively in the literature (Crespi and Miller, 1999; Masimir-
embwa et al., 1999). Care must be taken to ensure that the recombinant enzyme
selected has the appropriate kinetic parameters (Clarke, 1998) and that it is
compatible with the in vitro system in which it is studied.

Bioreactors
Bioreactors are growing cultures of cells expressing endogenous or heterologous
enzymes that can be used to metabolise drug compounds to their metabolites. The
advantages of bioreactors over conventional incubations are as follows.
Because the cells are growing, the culture can be sustained for longer periods of
time and therefore a higher yield of metabolites can be obtained. This is especially
useful if the rate of metabolite formation is slow.
Cultures can be scaled up to allow the extraction of enough low-abundance
metabolite for structural determination (NMR, MS).
Bioreactors can also perform reactions that are difficult to reproduce by synthetic
chemistry and are therefore invaluable for the production of authentic metabolite
standards and drug products. The most common recombinant bioreactor systems
are based on whole, baculovirus-infected insect cells and primary hepatocytes from

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Molecular biology 339

various species. The drive for hepatocyte bioreactor is the result of research into
artificial livers.
Site-directed mutagenesis (SDM)
The development of expression systems that can produce functional P450 protein
from a cDNA has allowed the manipulation of the proteins by site-directed
mutagenesis. This is a process where the sequence of the cDNA is deliberately
altered to change the protein structure of the encoded enzyme. This technique has
had three main applications in the field of drug metabolism.
Correction of cloning artefacts
During the late 1980s there was a race to isolate, sequence and publish the gene
sequences for P450 enzymes. In the rush to publish, mistakes were made and
retractions printed. Some of these mistakes were due to cloning errors which were
subsequently corrected by SDM.
A significant example of such a mix up concerned the cDNA for human
CYP2D6 where two forms existed, differing by a valine to methionine change at
amino acid residue 374. While the enzymes appeared to have the same catalytic
activity, studies with the probe substrate metoprolol and comparison to a human
liver bank showed that the 374 met form was the correct one and that the 374 val
form was probably a cloning artefact (Ellis et al., 1996). The validated gene
sequences are now available on the bioinformatics databases and a list of accession
numbers is periodically updated (Nelson et al., 1996).
Modelling of enzyme active sites
The ability to change individual residues has allowed extensive modelling of the
active site of enzymes. This has led to a greater understanding of the interaction
between enzymes and substrates, allowing rational drug design.
Producing soluble for crystallisation studies
In order to obtain good crystals, the protein must be reasonably soluble in a pure
form, a problem for mammalian P450s which tend to form insoluble precipitates
when removed from the membrane environment.
Recently, this problem has been overcome by a combination of removing the
membrane anchor region and mutation surface residues to make them more
polar. Using this approach Cosme and Johnson (2000) have managed to produce
crystals for the rabbit CYP2C5, the first mammalian P450 to be studied in
this way.
Immobilised P450s for biosensors
One application of expressed enzymes is that of biosensors, where an enzyme is
immobilised to a chip and activity determined electronically. The complex mem-
brane requirements of drug-metabolising enzymes (especially P450s) have hindered
progress in this area as a lipid membrane is difficult to build onto a chip.

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A novel solution has been proposed by Sligar and his group (Bayburt et al., 1998)
where the membrane is enclosed by a border of the HDL protein apolipoprotein A1.
This form ‘Nanodiscs’ of membrane ranging from 7 to 19 nm in diameter into
which P450 and reductase molecules can be inserted. These nanodiscs then provide
a means of attaching the enzymes to the surface of a chip.

Transgenic organisms
The development of transgenic technologies has allowed the study of gene function
in the whole organism (animal or plant). Such studies have taken the form of gene
addition (transgenics), gene deletions (knockouts), gene substitutions (knockins) or
nuclear transfer (cloning). The methodology and application of these methods to
DMPK is discussed below.
Gene additions: transgenics
Gene additions enable the study of the protein in a whole organism. This can take
the form of the over-expression of an endogenous protein or insertion of a gene from
another species into a host organism to study the effect against the endogenous
background. Such approaches have been used to study the effects of human P450
(Imaoka et al., 2001) in mice. Human P450s expressed in plants have been used in
bio-remuneration and detoxification.
Transgenics are produced by the method of pro-nuclear injection. Fertilised eggs are
removed from the donor animal and the pro-nucleus is injected with a vector
containing the gene of interest. The egg is implanted into a surrogate mother
and the transgene integrates randomly into the host genome. The resulting off-
spring will express the foreign gene in a site-dependent manner and not necessarily
at a level proportional to the number of copies integrated into the genome.
Gene deletions: knockouts
The ultimate way to inhibit an enzyme is to delete its expression from the test
organism. This can be done by utilising transgenic knockout techniques. This
method has been widely used to study the function of enzymes, nuclear hormone
receptors and transport proteins (Nebert and Duffy, 1997).
The method of choice for knockouts utilises embryonic stem cells. The gene for
the enzyme to be knocked out is identified in an embryonic stem cell line. Using
homologous recombination the gene is disrupted (usually by the insertion of a
selectable marker), and the modified stem cells are injected into an embryonic host.
The embryos are then allowed to mature into adult animals and the knockout is
assessed. Using selective breeding, a line of animals can be produced with both of the
genomic copies of the gene deleted. These animals can then be used in routine in vivo
and in vitro experiments to determine the effect of the knockout. This technique is
currently confined to mice due to the difficulty of culturing embryonic stem cells
from other organisms, although true rat ES cells may be available soon.

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Molecular biology 341

Gene substitution: knockins

Knockins use the same methodology as knockouts but instead of deleting the target
gene, it is replaced with a new gene. This new gene may be a mutated version of the
target gene, allowing the study of a polymorphism in an animal model. Another
application is to replace the host gene with one from another species which has
allowed the ‘humanisation’ of mice where the effects of the human gene can then be
studied in a mouse model (Xie et al., 2000).
Nuclear transfer
The field of nuclear transfer or ‘cloning’ is new and under the media spotlight. After
the arrival of Dolly the Sheep, Gene the Calf, Copy Cat and cloned mice in Hawaii,
the media and scientific press are full of the ethical and practical issues surrounding
this technology. The application of cloning to DMPK will develop, once the ethical
barriers have been resolved.

18.5 Induction or suppression?

1 8 . 5 . 1 B ACKG RO UND

Drug-metabolising enzymes can be modulated by the action of compounds on nuclear

receptors. This can lead to significant drug–drug interactions where the metabolism of
one drug is affected by the co-administration of a second. These changes can be
detected at various stages of the protein expression. Increases in protein amounts can
be detected by Western blotting, ELISA and proteomic techniques.
Increases in the mRNA levels (translational regulation) can be detected by
Northern blotting, RT-PCR, Riboprobes, RNA protection assays, differential gene
expression and Northern ELISA techniques.
Finally regulation at a transcriptional level can be detected with the use of
reporter gene constructs.
The majority of these techniques can be performed with commercially available
kits, as per the manufacturer’s protocols.

1 8 . 5 . 2 D ETERMIN A TIO N OF CHAN GE S I N P ROTEIN L EVEL S

Western blotting
Western blotting is a method of identifying a specific protein that has been separated
on an acrylamide gel, transferred to a nylon membrane and then detected using a
specific antibody. This antibody is then detected with a second antibody, labelled with
radioactivity or an enzyme that can be detected with a coloured substrate.

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342 Steve Hood

Western Blotting
+
Transfer to
Nylon Filter
BLOCK

WASH
–
PRIMARY
ANTIBODY

WASH

WASH SECONDARY
SUBSTRATE ANTIBODY

Section 11: New Technologies SH/DMDG/SC00/11

Figure 18.4 Western blotting.

As the amount of antibody binding to the membrane is proportional to the

amount of specific protein, this method can be semi-quantitative, providing that a
suitable analysis system is utilised to determine the band intensities (densitometer
or image analyser). This process is illustrated in Figure 18.4.

Enzyme-linked immunosorbent assay (ELISA)

ELISA is a variation of the Western blot where the protein to be determined is
immobilised to the surface of a microtitre well and then detected by the addition of
an antibody specific to the protein of interest. A second, enzyme-labelled, antibody
is then used to detect the first. Using a range of substrates which give coloured or
chemiluminescent products, the concentration of the protein can be determined to
some accuracy.
Due to the 96-well microtitre format of this assay it is possible to run several
samples and a standard curve simultaneously. Using automated plate washers and
readers, it is possible to get accurate readings of specific protein abundance. This
will give numerical data on the induction of enzymes at a protein level.
For more details see ‘Immunoassay: A Practical Guide’, B. Law (ed.) (1996),
ISBN 0–7484–0560–7.

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Molecular biology 343

Proteomics
Proteomics is a technique that combines gel electrophoresis and MS analysis to
identify changes in the levels of expression of all the proteins in a given biological
sample (Yoshida et al., 2001). This technique is currently being used to ‘fingerprint’
the expression changes in cells following exposure to toxic compounds in the hope
that a few ‘key’ proteins can be identified as markers of toxicity.
Proteins are separated from a biological matrix by two-dimensional gel electro-
phoresis. The proteins are first separated by their net charge using isoelectric
focussing on an immobilised pH gradient (IPG) strip. The strip is then placed in
contact with a polyacrylamide gel and the proteins are then separated by molecular
weight using standard PAGE methods. The resulting spots of protein can then be
sampled, analysed by TOF MS and identified against a database of known protein
structures. By comparing gels from control and induced cells, it is possible to
determine the changes in protein expression profiles.

1 8 . 5 . 3 D e te r m i n a ti on o f c h a n ge s i n m R N A levels

Polymerase chain reaction (PCR)

The polymerase chain reaction (PCR) has become a core technique for molecular
biologists and has allowed the development of several assays applicable to drug
metabolism. PCR allows the logarithmic amplification of a specific double-stranded
sequence of DNA from a background of other genes, proteins and cell debris.
The reaction is divided into three stages (see Figure 18.5):

Denaturation – The two strands in the target DNA are separated by heating the
reaction mixture to >95  C.
Annealing – Oligonucleotide primers bind to the ends of the template DNA. These
primers are designed to bind specifically to these sequences.
Elongation – The polymerase enzyme makes a copy of each strand using the primers
as a starting point until the target sequence has been duplicated. The cycle then
repeats with the newly copied versions of the target DNA acting as template for the
next cycle. As each cycle doubles the number of double-stranded template, the
product increases exponentially. Reactions are run for a minimum of 25 cycles
which results in 224 copies of each original template molecule.

This technique has been made possible by the discovery of thermostable poly-
merases that can withstand the repeated heating and cooling during the cycling of
the reaction. The reactions themselves are run in precision-controlled heating
blocks which can change temperature quickly and accurately and can be

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Figure 18.5 The PCR amplification process.

programmed for complex cycle profiles. For extensive methods, see PCR Protocols:
Methods in Molecular Biology, 15 (1993), B. White (ed.).

RT-PCR
As described previously, PCR requires double-stranded DNA to act as a template
for the amplification reaction and since mRNA is single stranded, it is therefore not
suitable for direct amplification by PCR. This problem can be overcome by the use
of a reverse-transcriptase enzyme which synthesises the complementary strand to
the mRNA, thus creating a double-stranded template for the PCR reaction.
This reaction has been optimised in a series of kits, and single polymerases have been
discovered that will perform the RT and PCR steps, thus making it a one-tube reaction.
Such a technique can be used to detect very low levels of a specific mRNA from a
tissue sample or cell culture. This technique has been used successfully in the
evaluation of gene therapy products.

Quantitative RT-PCR
The use of RT-PCR to determine changes in mRNA levels is becoming widespread
within drug metabolism and toxicology. The advantage of the technique is that
only a small amount of starting material (tissue or cell culture) is required to
provide the template for the reaction. This allows a higher throughput to be

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Molecular biology 345

achieved in induction studies (Morris and Davila, 1996). In most methods, the
changes in the amount of mRNA are normalised against a control mRNA,
whose expression level remains constant. These controls are usually the mRNA
for ‘housekeeping’ genes such as glyceraldehyde phosphate dehydrogenase
(GAPDH) or b-actin. It should be noted that the levels of expression of these
proteins may vary on xenobiotic challenge and must be closely monitored.
The original method of analysis for the RT-PCR products was to run them on a
gel and determine the staining intensity by desitometry on image analysis. This
approach was limited by the poor linearity of the stain intensity over a concentra-
tion range. Also, because the concentration of the products increases sigmoidaly,
linearity is lost at higher concentrations.
To overcome these problems, real-time measurement of product formation was
developed utilising fluorescent dyes to monitor the progress of the reaction. Two
approaches have been utilised.

Incorporation of intercollating dyes – Fluorescent dyes such as ethidium bromide and sybr
green only bind to double-stranded DNA and can therefore be used to follow the
production of the double-stranded products in a PCR reaction. This approach has been
used in the development of the LightCycler system by Roche Molecular Biochemicals.
Use of dye-labelled primers – An alternative approach, called Taqman, has been
developed by applied biosystems and utilises a dye-labelled primer which hybrid-
ises within the PCR product. One end of the primer is labelled with a fluorescent
dye and the other with a second dye which acts as a quencher. In its native state, the
light emitted from the first dye is absorbed by the second one, thus giving no
signal. During the PCR reaction the primer is degraded by the polymerase and the
reporter dye then dissociates from the quenching and is free to fluoresce.

Because both these techniques allow for real-time measurement of PCR product
formation, the rate of formation can be estimated from the linear part of the curve
(Figure 18.6). This leads to a more accurate determination of original copy number
and therefore mRNA induction. By comparing against a standard curve, it is
possible to calculate the amount of starting target mRNA in each sample.

Riboprobes
Riboprobes are short lengths (150–300 bp) of single-stranded RNA used as specific
probes for an mRNA of interest. The sequence of the cDNA of interest is examined by
computer to identify areas that are specific to that gene. Care must be taken in selecting
a region that has a sequence unique to the gene of interest. Such a region is usually
found in the 30 non-coding region (30 -NCR) where the homology between genes is low.
This region is then cloned (usually by PCR) into a vector whose multiple cloning
site is flanked by the promoters for RNA polymerase (most commercial vectors have

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346 Steve Hood

0.900
0.800
0.700
0.600
0.500
∆Rn

0.400
0.300
0.200
0.100
0.000
–0.100
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Cycle

Figure 18.6 A typical standard curve plot from a Taqman run showing the threshold line from which the cycle
number is calculated.

this configuration). Utilising an in vitro transcription kit, it is possible to produce

the riboprobe in both the sense and anti-sense strands. Incorporation of labelled
nucleotides in the polymerisation mix allows the production of labelled riboprobes.
These probes can then be hybridised to the target mRNA and thus used to detect
the presence of the mRNA in a sample (Rae et al., 2001).
Riboprobes may be used to determine mRNA concentration (see below) or used
in in situ hybridisation to locate the expression of the mRNA within a tissue of
interest. The latter approach has been used extensively to map the regulation of
gene expression in developmental biology.

RNase protection assay

The RNase protection assay is another method of quantifying the levels of a specific
mRNA extracted from a tissue (see Figure 18.7).
A riboprobe is designed to a short region of the target mRNA and is labelled
(usually with a 32 P dCTP). Total mRNA is extracted from the tissue and the probe
is added and allowed to hybridise to the target message. The mRNA is then treated
with RNAse, which selectively degrades single-stranded RNA. The mRNA/probe
hybrid is double stranded and is therefore not degraded in this digest. The digestion
products are run on an SDS-PAGE gel, imaged by autoradiography and the amount
of probe/mRNA hybrid is quantified against known standard. This allows a semi-
quantitative determination of the concentration of the target mRNA in the tissue of
interest (Rae et al., 2001). This technique requires the generation of highly labelled
probes and non-radioactive versions of this method have been developed to reduce
the amount of radioactivity utilised.

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Molecular biology 347

AAAAA
AAAAA
AAAAA
AAAAA
AAAAA AAAAA
AAAAA AAAAA
AAAAA AAAAA
Hybridise with
excess riboprobe
Digest with
RNAse A
cont [ mRNA]

_
AAAAA

AAAAA

Run on PAGE AAAAA

gel (and blot) AAAAA

AAAAA
+

Figure 18.7 The RNase protection assay.

Northern ELISA
Northern ELISA is a technique under development from Bohringer Manheim which
combines riboprobes and ELISA to give a more quantifiable readout. It is currently
experimental and little data exists on its utility for drug metabolism studies.

Differential gene expression (DGE)

Differential gene expression is a hybridisation technique that allows the comparison
of the mRNA levels in a specific tissue from different individuals or from different
tissues in the same individual. This enables the study of the modulation of mRNA
in induced or diseased animals. The developing technologies of radioactive and non-
radioactive imaging coupled with powerful image analysis software have enabled
thousands of genes to be studied in a single experiment. If the number of genes of
interest is small it is possible to simplify this procedure to a 96-well format. Efforts
are also underway to reduce the scale even more and by incorporation of microchip
technology, develop a ‘metabolism’ and ‘tox chip’.
The following example is for the study of genes regulated by an inducing
agent. A representative cDNA library for the tissue of interest (e.g. liver) is
gridded out in duplicate onto two nylon membranes. The livers from control and
induced animals are removed and mRNA isolated. The mRNA is converted to
cDNA and radiolabelled or fluorescently labelled nucleotides incorporated in the
process.

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348 Steve Hood

The labelled cDNA from the control liver is hybridised to the first membrane and
the cDNA from the induced liver to the other one. The intensity of the signal on each
of the gridded spots is compared between the control and induced samples, and the
imaging software identifies the spots where there is a difference in the intensity of the
signal. The identity of the induced gene can then be determined by referring back to
the original gridded library and sequencing the clone.
If the study is only interested in a specific subset of genes, smaller ‘focused’ arrays
can be produced containing only the cDNAs for the genes of interest. This allows for
simpler and quicker analysis and lends itself for adaptation to a microchip format. A
popular version of this is the Clonetech Atlas array that covers many drug-metabolising
enzymes and transcription factors (Rae et al., 2001).

1 8 . 5 . 4 D ETERMIN ATION OF CHAN GES I N GEN E T RAN S CRIPTI ON

Reporter gene assays

Reporter systems allow the study of gene regulation at a transcriptional level. The
promoter of the gene to be studied is cloned in front of the cDNA for a protein,
usually an enzyme, whose activity can be easily determined. The promoter/reporter
construct can then be introduced to a cell line or hepatocyte and used as a testy for
the induction of the target gene. Compounds that would normally modulate the
target gene will now have the same effect on the reporter gene and therefore such
modulation can be determined (Figure 18.8). Common reporter genes include
chloramphenecol acetyl transferase (CAT), secreted placental alkaline phosphatase
(SPAP), luciferase and green fluorescent protein (GFP).
For a reporter system to be representative of the true in vivo regulation of the gene the
complete promoter/enhancer region must be mapped and cloned. This is not a trivial

Promoter Intron Exon 3’UTR

DNA

Insert promoter
into vector
substrate
Test
compound reporter
enzyme product Assay

cDNA for
reporter gene

Appropriate cell line

Figure 18.8 Basis of reporter gene assay.

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Molecular biology 349

task as many genes are controlled by several regulatory elements, some of which may be
found at a significant distance from the promoter region. To date, therefore, reporter
systems have been confined to a few enzymes (CYP1A, 2B, 3A and 4A). The use of these
reporter genes to study enzyme induction is reviewed by Masimirembwa et al. (2001).

18.6 Population genetics and polymorphisms

1 8 . 6 . 1 I N TR O D U C TI O N T O P O L Y M O R P H I S M S

The phrase ‘no two people are the same’ has been confirmed by the Human Genome
Sequencing project where the structure and regulation of human genes are being
determined. During this project inter-individual differences in the gene sequences,
called as single nucleotide polymorphisms (SniPs for short), have been identified
and used to map the chromosomes on which they occur. By comparing the
distribution of these SNPs with known disease states, it is becoming possible to
find connections and possible causes of these problems.
Genetic polymorphisms in drug-metabolising enzymes and endogenous targets
have been shown to affect both the efficacy and safety of a variety of prescription
medicines. As the nucleic acid sequence of the human genome is determined, our
understanding of the importance of genetic polymorphisms on the pharmaco-
kinetics and pharmacodynamics of a drug is increasing. This knowledge has lead
to the development of simple genotyping tests that can help predict the disposition
and/or efficacy of a drug prior to administration. Genotyping technology will
impact the drug development process in the following ways:

1 Enable one to predict and understand the pharmacokinetic and pharmacody-

namic variability due to polymorphic drug-metabolising enzymes, receptors and
other molecular targets.
2 Used in disease diagnosis to confirm that the disease-causing mutation is present
in patient being treated; the relevance of polymorphisms in drug-metabolising
enzymes is reviewed by Nebert.

1 8 . 6 . 2 P OL YM ORPHISM S AND MIC RO HE TEROGE NEITY

It is important to note that not all phenotypic differences seen in the metabolism
of xenobiotics are due to genetic polymorphisms. Inter-individual variation due
to expression levels of the same ‘wild type’ gene can lead to significant differences
in the rates of metabolism for probe drugs. Such variation is described as micro-
heterogeneity and are less easy to assay for.

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1 8 . 6 . 3 P O L Y M O R P H I S M S I N D R U G - M E T A B O L I S I N G EN Z Y M E S
Modern genetic analysis is continually expanding the list of known polymor-
phism in drug-metabolising enzymes. Some of these mutations result in coding
changes while others are located in the regulatory regions of the gene and cause
differences in basal expression levels and inducibility. The most common poly-
morphic forms are:

CYP2C19
The polymorphisms of CYP2C19 and their detection are discussed in Goldstein and
de Morais (1994).

CYP2D6
The polymorphisms of CYP2D6 and their detection are discussed in Sachse et al.
(1997).

CYP2C9
The polymorphisms of CYP2C9 and their detection are discussed in Stubbins et al.
(1996).

CYP2E1
The polymorphisms of CYP2E1 and their detection are discussed in Powell et al.
(1998).

1 8 . 6 . 4 D ETEC TIO N OF POLYM ORPHISM S

Sequencing
Once an SNP has been identified, work is done to determine the frequency of this
polymorphism in a given population. This is usually done by obtaining genomic
DNA from hundreds of individuals and re-sequencing the part of the genomic
DNA containing the mutation. If the donors are part of a clinical trial panel or from
a liver bank it may be possible to determine if the SNP is associated with a specific
phenotypic change.
Once the SNP has been identified, it is possible to detect it with a variety of
analytical techniques and thus build a polymorphism screen. These techniques

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Molecular biology 351

include restriction fragment length polymorphism (RFLP) analysis, amplification

refractory mutation system (ARMS), single-strand conformation polymorphism
(SSCP) and the use of gene chips.
RFLP
RFLP analysis was the first technique developed to identify SNPs. It relies on the SNP
creating or removing a restriction enzyme site and is therefore not applicable for all
polymorphisms. A fragment spanning the SNP is amplified by PCR and then
digested with the appropriate enzyme. The digests are run on an agarose or poly-
acrylamide gel, and if an SNP is present in one or both of the alleles a differential
banding pattern will be observed. This technique is widely used in the identification
of the multiple CYP2D6 alleles (Daly et al., 1998).
ARMS
The ARMS is a PCR-based method which allows the detection of SNPs directly
from the genomic DNA. For a PCR reaction to be successful, the 30 -end of the
primer must be exactly the same as the target sequence. A mismatch in this position
will lead to a failure of the polymerase to elongate the new strand and no product
will be formed.
Two primers are made which match the DNA sequence immediately adjacent
to the SNP site with the last base of the primer complementary to the wild type or
the mutant sequence. If the target DNA contains the SNP of interest, a PCR
product will be obtained from the mutant primer but not the wild type. If there is
no polymorphism present then the pattern will be reversed. This technique has
been used successfully to identify polymorphisms in CYP2C9 (Stubbins et al.,
1996).

SSCP
SSCP relies on the physical properties of the PCR-amplified fragments. The rate of
migration of the single-stranded fragment through a polyacrylamide gel is determined
by its sequence, and by comparing the banding patterns under different gel conditions
it is possible to identify polymorphic variants. SSCP has been used to identify
polymorphisms in CYP2C9 (Stubbins et al., 1996).

Gene chips
Gene chips rely on determining the sequence of the target DNA by hybridisation to
fluorescently labelled oligonucleotides. The oligonucleotides are designed to bind the
DNA around the site of the SNP and can detect mismatches caused by polymorph-
isms. As it is possible to look for multiple SNP from a single DNA sample spotted
onto a chip, this method may offer a considerable increase in throughput and the
possibility of automated genotyping from a single drop of blood.

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18.7 References
Aoyama, T., Gonzalez, F.J. and Gelboin, H.V. (1989a) Human cDNA-expressed cytochrome
P450 IA2: mutagen activation and substrate specificity. Mol. Carcinog. 2(4), 192–198.
Aoyama, T., Yamano, S., Waxman, D.J., Lapenson, D.P., Meyer, U.A., Fischer, V., Tyndale, R.,
Inaba, T., Kalow, W. and Gelboin, H.V. (1989b) Cytochrome P-450 hPCN3, a novel
cytochrome P-450 IIIA gene product that is differentially expressed in adult human liver.
cDNA and deduced amino acid sequence and distinct specificities of cDNA-expressed
hPCN1 and hPCN3 for the metabolism of steroid hormones and cyclosporine. J. Biol. Chem.
264(18), 10388–10395.
Ball, S.E., Maurer, G., Zollinger, M., Ladona, M. and Vickers, A.E. (1992) Characterisation of
the cytochrome P-450 gene family responsible for the N-dealkylation of the ergot alkaloid
CQA 206–291 in humans. Drug Metab. Dispos. 20(1), 56–63.
Barnes, H.J. (1996) Maximising expression of eukaryotic cytochrome P450s in E. coli. Methods
Enzymol. 272, 3–14.
Bayburt, T.H., Carlson, J.W. and Sligar, S.G. (1998) Reconstitution and imaging of a membrane
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18.8 Bibliography
There are several good introductions to this subject. I have listed them in order of
complexity (and weight!).
Stryer, L. (1995) Biochemistry, 4th edn, USA, ISBN 0–7167–2009–4.
Alberts, B. et al. (1994) Molecular biology of the cell, 3rd edn, USA, Garland
publishing, ISBN 0–8153–1620–8.
Lewin, B., Genes, V. (1994) UK, Oxford University Press, ISBN0–19–854288–7.
Sambrook, J. (1989) Molecular cloning: A laboratory manual, 2nd edn, USA,
Cold Spring Harbor Laboratory Press, ISBN0–87969–309–6.

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