The impact of molecular neurobiology on psychopharmacology: from genes to drugs
About 150 years ago, Charles Darwin observed that ‘‘those who make many
species are the ‘splitters’, and those who make few are the ‘lumpers’’’.
Today, the ‘‘splitters’’ dominate research in the life sciences. Such
researchers can generate massive quantities of data on genes and their
networks, proteins and their pathways and the numerous cascades of
messenger molecules that ultimately result in a physiological response.
Technological progress in recent years has enabled the genome of species as
diverse as the nematode worm Caenorhabditis elegans and the fruit fly
Drosophila melanogaster to the mouse and man to be unravelled, thereby
opening up the possibility not only of identifying genes that are responsible
for physiological processes but also those that are aberrant and cause
genetically based diseases.
Few would deny the importance of such research, but the very success
of the ‘‘splitters’’ has had a seriously detrimental effect on the equally
important role of the ‘‘lumpers’’, who attempt to integrate the
molecular/cellular approach with the behavioural/psychological consequences.
As a consequence, the ‘‘lumpers’’ are becoming a threatened
species of researchers. There are several reasons for this, not the least of
which is the widespread opposition to vivisection and the lack of
training in behavioural pharmacology in university courses. As a
consequence, research (and funding for behavioural research) has
declined in prestige. This has had an adverse impact not only in areas
of basic life science research but also in the pharmaceutical industry
where the ultimate validation of the therapeutic potential of a new
molecule depends on behavioural pharmacology. As a senior neuropharmacologist
has recently remarked ‘‘Many can genotype but few can
phenotype’’.
Despite this unfortunate disparity between molecular neurobiology and
behavioural pharmacology, it is essential that the neuropharmacologist and
biological psychiatrist are fully conversant with the basic concepts of the
subject in order to appreciate both its success and limitations.
To understand the basis of cloning, it isnecessary to consider how bacteria have evolved to resist infection by
external sources of genetic material. It has long been recognized that if a
virus could infect one strain of bacteria, it could then also infect other
bacteria of the same strain but not those of a different strain. Thus virus
infection was shown to be restricted to a particular strain, a restriction now
known to be due to two classes of enzyme, namely the methylases, which
modify bacterial DNA marking them as ‘‘self’’, and the destruction
enzymes, which act as molecular ‘‘scissors’’ and can destroy foreign DNA.
Restriction enzymes are sequence-specific in that they cut DNA at specific
locations along the nucleotide chain. While some of these enzymes yield
‘‘blunt’’ ends to the resulting DNA fragment, others make staggered cuts in
the DNA chain to produce ‘‘sticky’’ ends. Over 250 restriction enzymes are
now commercially available.
Cloning would not be possible without restriction enzymes. DNA chains
with a ‘‘sticky’’ end act like molecular ‘‘Velcro’’, thereby enabling two
pieces of DNA with complementary nucleotide sequences to be joined
together. The linking of the DNA strands is brought about by the enzyme
DNAligase which permanently joins the assembled DNA sequences with
covalent bonds, thereby producing a recombinant DNA molecule.
The next stage is to ensure that the recombinant DNA molecule is
copied by the enzymes which synthesize nucleic acids. These DNA and
RNA polymerases synthesize an exact copy of either DNA or RNA from
a pre-existing molecule. In this way the DNA polymerase duplicates the
chromosome before each cell division such that each daughter cell will
have a complete set of genetic instructions which are then passed to the
newly formed RNA by RNA polymerase. While both DNA and RNA
polymerase require a preformed DNA template, some viruses (such as
HIV) have an RNA genome. To duplicate that genome, and incorporate
it into a bacterial or mammalian cell, the viruses encode a reverse
transcriptase enzyme which produces a DNA copy from an RNA
template.
Thermostable DNA polymerases have now been produced for
polymerase chain reaction (PCR) studies in which specific segments of
the DNA molecule can be mass produced from minute quantities of
material. RNA polymerases are then used to create RNA transcripts from
cloned genes in vitro. Reverse transcriptases have their specific uses in
molecular biology. These enzymes are used to form ‘‘cDNA libraries’’
which are batteries of molecules each one representing a single gene
expression. Such DNA libraries can then be analysed to determine which
genes are active under different conditions and in different tissues. cDNA
libraries are now used experimentally in microarray assemblies to detect
gene changes following drug treatment.
In a typical experimental situation, the gene of interest is incorporated
into a plasmid, which is a natural vector used by either a bacterium or other
cell type. To transfer the DNA fragment of a gene, the plasmids are digested
with one or two restriction enzymes and the desired fragment joined into a
single DNA recombinant molecule using DNA ligase. To express the new
gene in vitro, the plasmid containing the recombinant DNA is then
incubated with an RNA polymerase to form new RNA which is then
used to programme an in vitro system which translates the information
necessary for the synthesis of a new protein.
The foregoing is only intended to give a brief overview of the
mechanisms behind cloning. So far, the impact on diseases in man has
been limited to experimental approaches to the treatment of cystic
fibrosis and rare conditions in which a recessive gene is responsible.
However, cloning techniques have provided important information in
producing animals, usually mice, which have been manipulated to
express or remove genes that are implicated in psychiatric disorders.
Such ‘‘knock-out’’ and ‘‘knock-in’’ mice now provide important
information in which specific genes can be studied for their effects
on behaviour, which may ultimately be an important contribution to
understanding the genetic basis of psychiatric and neurological
diseases.
About 150 years ago, Charles Darwin observed that ‘‘those who make many
species are the ‘splitters’, and those who make few are the ‘lumpers’’’.
Today, the ‘‘splitters’’ dominate research in the life sciences. Such
researchers can generate massive quantities of data on genes and their
networks, proteins and their pathways and the numerous cascades of
messenger molecules that ultimately result in a physiological response.
Technological progress in recent years has enabled the genome of species as
diverse as the nematode worm Caenorhabditis elegans and the fruit fly
Drosophila melanogaster to the mouse and man to be unravelled, thereby
opening up the possibility not only of identifying genes that are responsible
for physiological processes but also those that are aberrant and cause
genetically based diseases.
Few would deny the importance of such research, but the very success
of the ‘‘splitters’’ has had a seriously detrimental effect on the equally
important role of the ‘‘lumpers’’, who attempt to integrate the
molecular/cellular approach with the behavioural/psychological consequences.
As a consequence, the ‘‘lumpers’’ are becoming a threatened
species of researchers. There are several reasons for this, not the least of
which is the widespread opposition to vivisection and the lack of
training in behavioural pharmacology in university courses. As a
consequence, research (and funding for behavioural research) has
declined in prestige. This has had an adverse impact not only in areas
of basic life science research but also in the pharmaceutical industry
where the ultimate validation of the therapeutic potential of a new
molecule depends on behavioural pharmacology. As a senior neuropharmacologist
has recently remarked ‘‘Many can genotype but few can
phenotype’’.
Despite this unfortunate disparity between molecular neurobiology and
behavioural pharmacology, it is essential that the neuropharmacologist and
biological psychiatrist are fully conversant with the basic concepts of the
subject in order to appreciate both its success and limitations.
To understand the basis of cloning, it isnecessary to consider how bacteria have evolved to resist infection by
external sources of genetic material. It has long been recognized that if a
virus could infect one strain of bacteria, it could then also infect other
bacteria of the same strain but not those of a different strain. Thus virus
infection was shown to be restricted to a particular strain, a restriction now
known to be due to two classes of enzyme, namely the methylases, which
modify bacterial DNA marking them as ‘‘self’’, and the destruction
enzymes, which act as molecular ‘‘scissors’’ and can destroy foreign DNA.
Restriction enzymes are sequence-specific in that they cut DNA at specific
locations along the nucleotide chain. While some of these enzymes yield
‘‘blunt’’ ends to the resulting DNA fragment, others make staggered cuts in
the DNA chain to produce ‘‘sticky’’ ends. Over 250 restriction enzymes are
now commercially available.
Cloning would not be possible without restriction enzymes. DNA chains
with a ‘‘sticky’’ end act like molecular ‘‘Velcro’’, thereby enabling two
pieces of DNA with complementary nucleotide sequences to be joined
together. The linking of the DNA strands is brought about by the enzyme
DNAligase which permanently joins the assembled DNA sequences with
covalent bonds, thereby producing a recombinant DNA molecule.
The next stage is to ensure that the recombinant DNA molecule is
copied by the enzymes which synthesize nucleic acids. These DNA and
RNA polymerases synthesize an exact copy of either DNA or RNA from
a pre-existing molecule. In this way the DNA polymerase duplicates the
chromosome before each cell division such that each daughter cell will
have a complete set of genetic instructions which are then passed to the
newly formed RNA by RNA polymerase. While both DNA and RNA
polymerase require a preformed DNA template, some viruses (such as
HIV) have an RNA genome. To duplicate that genome, and incorporate
it into a bacterial or mammalian cell, the viruses encode a reverse
transcriptase enzyme which produces a DNA copy from an RNA
template.
Thermostable DNA polymerases have now been produced for
polymerase chain reaction (PCR) studies in which specific segments of
the DNA molecule can be mass produced from minute quantities of
material. RNA polymerases are then used to create RNA transcripts from
cloned genes in vitro. Reverse transcriptases have their specific uses in
molecular biology. These enzymes are used to form ‘‘cDNA libraries’’
which are batteries of molecules each one representing a single gene
expression. Such DNA libraries can then be analysed to determine which
genes are active under different conditions and in different tissues. cDNA
libraries are now used experimentally in microarray assemblies to detect
gene changes following drug treatment.
In a typical experimental situation, the gene of interest is incorporated
into a plasmid, which is a natural vector used by either a bacterium or other
cell type. To transfer the DNA fragment of a gene, the plasmids are digested
with one or two restriction enzymes and the desired fragment joined into a
single DNA recombinant molecule using DNA ligase. To express the new
gene in vitro, the plasmid containing the recombinant DNA is then
incubated with an RNA polymerase to form new RNA which is then
used to programme an in vitro system which translates the information
necessary for the synthesis of a new protein.
The foregoing is only intended to give a brief overview of the
mechanisms behind cloning. So far, the impact on diseases in man has
been limited to experimental approaches to the treatment of cystic
fibrosis and rare conditions in which a recessive gene is responsible.
However, cloning techniques have provided important information in
producing animals, usually mice, which have been manipulated to
express or remove genes that are implicated in psychiatric disorders.
Such ‘‘knock-out’’ and ‘‘knock-in’’ mice now provide important
information in which specific genes can be studied for their effects
on behaviour, which may ultimately be an important contribution to
understanding the genetic basis of psychiatric and neurological
diseases.
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