A.5 NeurophArmACology (Ahl)
acting neurotransmitters can modulate ast synaptic transmission or Endorphins
relatively long periods o time.
Endorphins can act as
Memory and learning painkillers.
Memory and learning involve changes in neurons caused Pain receptors in the skin and other
by slow-acting neurotransmitters. parts o the body detect stimuli
such as the chemical substances
Psychologists have studied learning and memory or decades but in a bees sting, excessive heat
it is only relatively recently that neurobiologists have been able or the puncturing o skin by a
to study these processes at the level o the synapse. Slow-acting hypodermic needle. These receptors
neurotransmitters (neuromodulators) have a role in memory and are the endings o sensory neurons
learning. They cause the release o secondary messengers inside that convey impulses to the
post-synaptic neurons that can promote synaptic transmission by central nervous system. When
mechanisms such as an increase in the number o receptors in the impulses reach sensory areas o
post-synaptic membrane or chemical modifcation o these receptors the cerebral cortex we experience
to increase the rate o ion movements when neurotransmitter binds. the sensation o pain. Endorphins
are oligopeptides that are secreted
The secondary messengers can persist or days and cause what is by the pituitary gland and act as
known as long-term potentiation ( LTP) . This may be central to natural painkillers, blocking eelings
the synaptic plasticity that is necessary or memory and learning. o pain. They bind to receptors in
Even longer-term memories may be due to a remodelling o the synapses in the pathways used in
synaptic connections between neurons. The learning o new skills the perception o pain, inhibiting
has been shown to be linked to the ormation o new synapses in synaptic transmission and
the hippocampus and elsewhere in the brain. preventing the pain being elt.
Psychoactive drugs
Psychoactive drugs afect the brain by either increasing or
decreasing post-synaptic transmission.
The brain has many synapses, perhaps as many as 1 016 in children.
These synapses vary in their organization and use a wide variety o
neurotransmitters. Over a hundred dierent brain neurotransmitters are
known. Psychoactive drugs aect the brain and personality by altering
the unctioning o some o these synapses. S ome drugs are excitatory,
because they increase post-synaptic transmission. Others are inhibitory
because they decrease it.
Examples o excitatory drugs:
Nicotine contained in cigarettes and other orms o tobacco, derived
rom the plant Nicotiana tabacum.
C ocaine extracted rom the leaves o a Peruvian plant, Erythroxylon coca.
Amphetamines, a group o artifcially synthesized compounds.
Examples o inhibitory drugs:
B enzodiazepines, a group o compounds including Valium that are
synthesized artifcially.
Alcohol in the orm o ethanol, obtained by ermentation using yeast.
Tetrahydrocannabinol ( THC ) obtained rom the leaves o the
Cannabis sativa plant.
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A NEUROBIOLOGY AND BEHAVIOUR
Ecstasy
Evaluation o data showing the impact o MDMA (ecstasy) on serotonin and
dopamine metabolism in the brain.
data-base questions: Efects o ecstasy on the striatum
The graphs in fgure 2 show the results o an 2 Discuss the evidence rom the data or the
experiment in which mice were treated with hypothesis that MDMA has a greater eect
MDMA (ecstasy) and levels o dopamine and on serotonin level than dopamine level in
serotonin were measured in the striatum o the wild-type mice. [3]
their brains. Two doses o MD MA were used 3 a) Distinguish between the results or the
and also saline (no MDMA) . Wild-type mice wild-type mice and the DAT-KO mice. [2]
were used and also three strains o knockout
mice that lacked genes or making the dopamine b) Discuss whether these dierences are
transporter protein ( D AT-KO) , the serotonin statistically signifcant or not. [2]
transporter ( SERT-KO) or both transporters 4 Distinguish between the results or the
( D AT/SERT-KO ) . The graphs show the levels D AT-KO mice and the S ERT-KO mice. [2 ]
o dopamine and serotonin in the three-hour
period ater MDMA had been administered. 5 Explain the results or the D AT/S ERT- KO
Questions mice. [2]
1 Describe the trends in dopamine level 6 Suggest one beneft o using knockout mice
wild-type mice in the three hours ater
in this experiment. [1 ]
administration o 1 0 mg o MDMA. [3]
Wild, 3 mg
500 4000 DAT-KO, 3 mg
SERT-KO, 3 mg
dopamine (% of basal level) 3500 DAT/SERT-KO, 3 mg
serotonin (% of basal level)
400 Wild, 10 mg
2500 DAT-KO, 10 mg
SERT-KO, 10 mg
300 2000 DAT/SERT-KO, 10 mg
200 1500
1000
100
500
0 0
-20 0 20 40 60 80 100 120 140 160 180 -20 0 20 40 60 80 100 120 140 160 180
Figure 2 time (min) time (min)
Reference: Hagino et al, Efects o MDMA on Extracellular Dopamine and Serotonin Levels in Mice Lacking Dopamine and/or Serotonin
Transporters, Curr. Neuropharmacol. 2011 March; 9(1) : 9195.
Anaesthetics
Anaesthetics act by interering with neural transmission
between areas o sensory perception and the CNS.
Anaesthetics cause a reversible loss o sensation in part or all o the
body. Local anaesthetics cause an area o the body to become numb,
or example the gums and teeth during a dental procedure. General
anaesthetics cause unconsciousness and thereore a total lack o sensation.
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A.5 NeurophArmACology (Ahl)
Anaesthetics are chemically varied and work in a variety of ways. Many
of them affect more than just the sense organs and can also inhibit signals
to motor neurons and other parts of the nervous system so they should
only ever be administered by highly trained medical practitioners.
Anaesthetics and awareness
The efect o anaesthetics on awareness.
A patient who has been given a general anaesthetic they have not been given a high enough dose
normally has no awareness of the surgical or other of anaesthetic. The patient may or may not
procedures that they are undergoing because they feel pain. The risk of awareness is highest in
are totally unconscious. There are some procedures operations such as emergency caesarean sections
where it is either not necessary or is undesirable for in which it is best for the mother and child
the patient to be unconscious. For example patients for the dose of anaesthetic to be minimized,
are kept partially conscious during some operations although in these procedures a spinal block is
to remove brain tumours, so that the effects on the almost always now used rather than a general
brain can be monitored. anaesthetic, so the patient is awake and
breathing is normal, but pain sensation cannot
There have been some cases of patients retaining get beyond the spinal cord.
some awareness during operations, when
Drug testing
Assessing risk associated with scientic research: patient
advocates will oten press or the speeding up odrug
approval processes, encouraging more tolerance orisk.
There are strict protocols for testing new drugs with several phases that
establish two things an appropriate dose and route of administration
that make the drug effective, and that its side-effects are minor and
infrequent enough for the drug to be regarded as safe. These tests take
many years to complete but approval for the introduction of a new drug
is only given once the tests have been rigorously carried out. There
have been some trials where the difference between the control group
of patients given a placebo and the group given the new drug is so
great that it seems unethical to deny the control group the treatment.
It therefore seems reasonable to abandon the trials and introduce the
drug immediately. The danger of this policy is that harmful side-effects
may only then be discovered when large numbers of patients have been
given the new drug.
There have been some cases where groups of patients have
campaigned for a new drug to be introduced before it has been
fully tested. This may be acceptable with terminal diseases such
as AIDS or certain forms of heart disease where the patient may
regard any level of risk acceptable given the certainty of death
without treatment. It is unlikely to be acceptable with non-critical
illnesses where the risks from using a drug that has not been fully
tested are too great compared with the risks associated with the
disease remaining untreated.
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A NEUROBIOLOGY AND BEHAVIOUR
Figure 3 Drug enforcement Stimulant drugs
measures near a school
Stimulant drugs mimic the stimulation provided by the
sympathetic nervous system.
Stimulants are drugs that promote the activity o the nervous system.
They make a person more alert, energetic and sel-confdent. They also
increase heart rate, blood pressure and body temperature. The eects
o stimulant drugs match those o the sympathetic nervous system.
This is because stimulant drugs act by a variety o mechanisms to make
the body respond as though it had been naturally stimulated by the
sympathetic nervous system.
Some mild stimulants are present in oods and drinks, or example
caeine in tea and coee and theobromine in chocolate. Doctors
sometimes prescribe stronger stimulants to treat conditions such as
clinical depression and narcolepsy. S timulant drugs are also sometimes
used against medical advice. Examples include cocaine, amphetamines
and nicotine in cigarettes.
Examples of stimulants and sedatives
Efects on the nervous system o two stimulants and two sedatives.
Pramipexole mimics dopamine and binds to GABA (-amino butyric acid) is an inhibitory
dopamine receptors in post-synaptic membranes at neurotransmitter and when it binds to its
dopaminergic synapses. Whereas some drugs that receptor a chloride channel opens, causing
mimic neurotransmitters are antagonists because hyperpolarization o the post-synaptic neuron
they block synaptic transmission, pramipexole by entry o chloride ions. When diazepam is
is an agonist because it has the same eects as bound to the receptor the chloride ions enter at
dopamine when it binds. Pramipexole is used a greater rate, inhibiting nerve impulses in the
during the early stages o Parkinsons disease to post-synaptic neuron. Diazapam is thereore a
help to reduce the eects o insufcient dopamine sedative. It can reduce anxiety, panic attacks
secretion that characterize this disease. It has also and insomnia and it is also sometimes used as a
sometimes been used as an anti-depressant. muscle relaxant.
Cocaine also acts at synapses that use dopamine THC ( Tetrahydrocannabinol) is present in
as a neurotransmitter. It binds to dopamine cannabis. It binds to cannabinoid receptors
reuptake transporters, which are membrane in pre-synaptic membranes. Binding inhibits
proteins that pump dopamine back into the the release o neurotransmitters that cause
pre-synaptic neuron. Because cocaine blocks excitation o post-synaptic neurons. THC is
these transporters, dopamine builds up in the thereore an inhibitory psychoactive drug and
synaptic clet and the post-synaptic neuron a sedative. Cannabinoid receptors are ound in
is continuously excited. Cocaine is thereore synapses in various parts o the brain, including
an excitatory psychoactive drug that gives the cerebellum, hippocampus and cerebral
eelings o euphoria that are not related to any hemispheres. The main eects o THC are
particular activity. disruption o psychomotor behaviour, short-
term memory impairment, intoxication and
D iazepam ( Valium) binds to an allosteric site on stimulation o appetite.
GABA receptors in post-synaptic membranes.
546
A.5 NeurophArmACology (Ahl)
Drug addiction Figure 4 Alcohol is an addictive drug but is
legal in many counties
Addiction can be afected by genetic predisposition, social
environment and dopamine secretion.
The American Psychiatric Association has defned addiction as:
a chronically relapsing disorder that is characterized by three main elements:
(a) compulsion to seek and take the drug, (b) loss of control in limiting intake and
(c) emergence of a negative emotional state when access to the drug is prevented.
Only certain drugs cause addiction and usually repeated use over a
prolonged period o time is needed. With a ew drugs, addiction can
develop more rapidly. The causes o addiction are clearly not simple and
three areas need to be considered.
1 Some people seem much more vulnerable to addiction than others
because o their genes. This is known as genetic predisposition.
One example is the gene, DRD2, which codes or the dopamine
receptor protein. There are multiple alleles o this gene and a recent
study showed that people with one or more copies o the A1 allele
consumed less alcohol than those homozygous or the A2 allele.
2 Addiction is more prevalent in some parts o society than others
because the social environment greatly aects the likelihood o
taking drugs and becoming addicted. Peer pressure, poverty and
social deprivation, traumatic lie experiences and mental health
problems all contribute. Cultural traditions are very important and
help to explain why dierent drugs cause problems in dierent parts
o the world.
3 Many addictive drugs, including opiates, cocaine, nicotine and
alcohol aect dopamine secreting synapses. Dopamine secretion is
associated with eelings o well-being and pleasure. Addictive drugs
cause prolonged periods with high dopamine levels in the brain.
This is so attractive to the drug user that they fnd it very difcult
to abstain.
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A.6 etoogy (Ahl)
Understanding Applications
Ethology is the study o animal behaviour in Migratory behaviour in blackcaps as an example
natural conditions. o the genetic basis o behaviour and its change
by natural selection.
Natural selection can change the requency o
observed animal behaviour. Blood sharing in vampire bats as an example o
the evolution o altruistic behaviour by natural
Behaviour that increases the chances o selection.
survival and reproduction will become more
prevalent in a population. Foraging behaviour in shore crabs as an
example o increasing chances o survival by
Learned behaviour can spread through a optimal prey choice.
population or be lost rom it more rapidly than
innate behaviour. Breeding strategies o hooknoses and jacks
in coho salmon populations as an example o
Nature of science behaviour afecting chances o survival and
reproduction.
Testing a hypothesis: experiments to test
hypotheses on the migratory behaviour o Courtship in birds o paradise as an example o
blackcaps have been carried out. mate selection.
Synchronized oestrus in emale lions in a
pride as an example o innate behaviour
that increases the chances o survival and
reproduction o ofspring.
Feeding on cream rom milk bottles in blue tits
as an example o the development and loss o
learned behaviour.
Ethology
Ethology is the study o animal behaviour in natural
conditions.
Animals are adapted to their natural habitat in their behaviour. I we
remove them rom this habitat and place them in a zoo or laboratory,
animals may not behave normally because they may not receive
the same stimuli as in their natural habitat. For this reason it is best
whenever possible to carry out research into animal behaviour in their
natural habitat rather than in an artifcial environment. The study
o the actions and habits o animals in their natural environment is
called ethology.
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A.6 ethology (Ahl)
Natural selection and animal behaviour
Natural selection can change the requency o observed
animal behaviour.
Natural selection is the theme that runs through the whole o
modern biology, including ethology. It adapts species to all aspects
o their environment. Adaptation extends over the whole range o
animal characteristics, rom the structure o a single molecule such as
hemoglobin to the patterns o behaviour in a species.
Animal behaviour has been observed to change rapidly in some cases.
House fnches Carpodacus mexicanus are an example. In C aliornia the
native population is sedentary the birds remain in the same area
throughout the year. A small number were illegally released in the 1 940s
in New York C ity and spread though the eastern United S tates, and
within twenty years migratory behaviour was observed. The requency
o this behaviour rose to more than 50% o the population, presumably
as a result o natural selection.
The mechanism of natural selection
Behaviour that increases the chances o survival and
reproduction will become more prevalent in a population.
Natural selection works in same way or animal behaviour as or other
biological characteristics. Individuals with the best-adapted actions and
responses to the environment are most likely to survive and produce
ospring. I behaviour is genetically determined, rather than learned, it
can be inherited by ospring.
The breeding season o the great tit Parus major illustrates how behaviour
evolves by natural selection, oten as a response to environmental
changes. This bird lives in woodland and eeds its young on caterpillars
and other insects. The availability o this ood rises to a peak in spring
soon ater the new leaves on trees have grown. Due to global warming,
the time o peak availability has become earlier. The timing o nesting
and egg laying varies within narrow limits within the population.
Researchers have shown that birds that lay their eggs a ew days earlier
than the mean date have more success in rearing young. According to
natural selection, the mean date o egg laying should evolve to be earlier
and researchers ound this prediction ulflled.
Breeding strategies in salmon
Breeding strategies o hooknoses and jacks in coho salmon populations as an
example o behaviour afecting chances o survival and reproduction.
C oho salmon Oncorhynchus kisutch breed in rivers The adults die ater breeding and the young live
that discharge into the North Pacifc Ocean, or about a year in the river and then migrate to
including those on the west coast o North America. the ocean where they remain or several years
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A NEUROBIOLOGY AND BEHAVIOUR
beore returning to spawn. There are two breeding hooknoses are unlikely to sneak up on a emale
strategies among males. Hooknoses fght each other without being noticed so they must fght other
or access to emales laying eggs, with the winner hooknoses and end o jacks i they are to be
shedding sperm over the eggs to ertilize them. successul in breeding.
Jacks usually avoid fghts and instead sneak up on
emales and attempt to shed sperm over their eggs Figure 1 Brown bear catching salmon as they swim
beore being noticed. upstream to breed
Obervations on individually identifed fsh,
usually identifed by a tag, show that whether
a male becomes a jack or a hooknose depends
on his growth rate. Males that grow rapidly
are able to return to breed two years ater they
were spawned and are jacks. Males that grow
less rapidly remain in the ocean or one year
longer, but are then signifcantly larger and are
hooknoses. The smaller jacks are more likely
to reproduce by the sneaking strategy than
by fghting the larger hooknoses. The larger
Synchronized oestrus
Synchronized oestrus in emale lions in a pride as an example o innate behaviour
that increases the chances o survival and reproduction o ofspring.
Female lions remain in the group (pride) into oestrus at the same time. This behaviour has
which they were born, but male lions are several advantages: the emales have their cubs
expelled rom the pride when they are about at the same time so are all lactating while the
three years old. Males can only breed i, when cubs are suckling, so they can suckle each others
ully grown adults, they overcome the dominant cubs when they are hunting, increasing the cubs
male in another pride by fghting. Within two chance o survival. Also a group o male cubs
or three years o taking over a pride o emales, o the same age are ready to leave the pride at
the breeding male is likely to be replaced by a the same time so can compete or dominance o
younger rival. When a new dominant male takes another pride more eectively.
over a pride he may kill all the suckling cubs,
thus making the emales come into oestrus more
quickly so the male can then mate with them to
ather his own cubs. Females protect their cubs
rom marauding males, sometimes leading to
ferce fghts, but accept his sexual advances ater
he has taken over the pride. Sometimes two or
more closely related young males together fght
or dominance o another group. This increases
their chance o success, especially i they are
fghting a single dominant male.
Females can only breed when they come into Figure 2 Lions in a group known as a pride
oestrus. All emales in a pride tend to come into
550
A.6 ethology (Ahl)
Blackcap migration
Migratory behaviour in blackcaps as an example of the genetic basis of behaviour
and its change by natural selection.
The blackcap Sylvia atricapilla breeds during There are several possible reasons or this change
the northern summer. Until relatively recently, in migration behaviour. Global warming has led
populations o blackcaps that breed in Central to winters being warmer in Britain so the long
Europe including Germany almost all migrated migration to S pain is not necessary. Many people
to S pain and Portugal or the winter, where the in Britain eed wild birds in winter which may
weather is warmer and the availability o ood acilitate survival o overwintering blackcaps more
is greater. D uring the second hal o the 2 0th than in Spain. In winter the minimum day length
century a ew blackcaps rom the population in in Britain is shorter than in Spain, which may
Germany were ound to be migrating to Britain prompt earlier migration to breeding grounds.
and Ireland instead. The numbers o blackcaps Blackcaps that arrive earlier take the best
overwintering in Britain rose rapidly to more territories another advantage o overwintering
than 1 0%. in Britain.
Experiments with migrating blackcaps
Testing a hypothesis: experiments to test hypotheses on the migratory behaviour
of blackcaps have been carried out.
In ethology as in other branches o science it is migratory stimuli in the same way as their
essential to test hypotheses and either obtain parents, indicating that the direction o
evidence or them or prove them to be alse. migration is genetically determined, and can
The adaptive value o behaviour patterns have thus be subject to long-term evolutionary
sometimes been assumed without evidence. change under natural selection.
These accounts o evolution are known as Just
So Stories ater Rudyard Kiplings childrens FPO
story book. However intuitively obvious a Brita in< 8 3 9 2 1 1 _p h A. 6 . 5 >
hypothesis about the evolution o a behaviour
pattern, it is only a just so story until tested. ( wi nter)
Germany
Hypotheses about evolutionary changes in
blackcap migration have been rigorously tested. (summer)
For example, the hypothesis that the direction
o migration is genetically determined has Spain
been tested. Eggs were collected in Germany ( winter)
rom parent birds that had migrated to Britain
in the previous winter and rom parents who Figure 3 Migration of blackcaps
had migrated to Spain. The young were reared
without their parents so that they could not
learn rom them and when they migrated the
direction was recorded. Birds whose parents
had migrated to Britain tended to fy west,
wherever they were reared, and birds whose
parents had migrated to Spain tended to fy
south-west. They thereore responded to
551
A NEUROBIOLOGY AND BEHAVIOUR
Vampire bats
Blood sharing in vampire bats as an example of the evolution of altruistic
behaviour by natural selection.
Female vampire bats Desmodus rotundus live in on o genes o the altruistic animal. Blood sharing
colonies o 81 2 individuals, with the same is an example o reciprocal altruism. Individual A
individuals roosting together or several years. gains a beneft rom giving blood to Individual B
Their diet is about 25ml o vertebrate blood, because Individual B survives and can share blood
usually mammalian, each night. I a bat ails to on a later night i Individual A ails to eed. It
eed or two or three consecutive nights they only occurs in stable groups o emales that roost
risk death rom starvation. However, this rarely together regularly and as it aids the chances o
happens because when the bats return to the survival and reproduction o all o the members o
roost at the end o the night, those that have ed such groups, natural selection avours it.
regurgitate blood or those that have not.
This behaviour pattern is a rare example o
altruism. It ulfls two necessary criteria:
there may be siblings or mothers with
daughters in a group but tests have shown that
there are also unrelated emales who also share
blood, so blood sharing is not kin-selection;
giving blood to an individual who has not ed
incurs a cost to the giver because some o their
daily diet is lost, so blood sharing is not merely
cooperation it is genuine altruism.
The evolution o altruism is an interesting Figure 4 Vampire bats show reciprocal altruism by
conundrum: we might not expect natural blood sharing
selection to promote the evolution o behaviour
that incurs a cost, because it should reduce the
chances o survival, reproduction and the passing
Foraging in shore crabs
Foraging behaviour in shore crabs as an example of increasing chances of
survival by optimal prey choice.
Foraging is searching or ood. Animals (a) 1.5
must decide what type o prey to search 1.0
(b) 50
or and how to fnd it. Studies have shown protability/J s-1 40
per cent of diet 30
that the prey chosen by animals tends to
be the type that gives the highest rate o
energy return. For example, the shore crab 0.5 20
Carcinus moenas preers to eat mussels o 10
intermediate size when presented in an 1-0 2-0 3-0 4-0 0
aquarium with equal numbers o each size, size of mussel/cm 1-0 1-5 2-0 2-5 3-0
as shown in the bar chart in fgure 5. The
size of mussel/cm
graph in fgure 5 shows that mussels o Figure 5 Proftability in Joules per second and percentage in diet o
intermediate size are the most proftable mussels o dierent size
in terms o the energy yield per second o
time spent breaking open the shells.
552
A.6 ethology (Ahl)
Courtship in birds of paradise
Courtship in birds o paradise as an example o mate selection.
Some animals have anatomical eatures that seem used by birds o paradise and biologists have long
to the human eye to be excessive, or example speculated on the reasons or exaggerated traits.
the tail eathers o the peacock. Other animals
have behaviour patterns that seem bizarre. The Darwin explained them in terms o mate
plumage and courtship displays o male birds o selection emales preer to mate with males that
paradise are examples o both o these types o have exaggerated traits. The reason may be that
exaggerated trait. There are about orty species these traits indicate overall tness. I a bird o
o bird o paradise living on New Guinea and paradise has enough energy to grow and maintain
other nearby islands. The males have very showy the elaborate plumage and repeatedly to carry
plumage with bright coloration and elongated or out very vigorous courtship displays it indicates
elaborate tail eathers that are o no use in fying. that the male must have ed eciently. I it can
The emales, which build the nest, incubate the survive in the rainorest with the encumbrance
eggs and rear the young, are relatively drab. o its tail eathers and with bright plumage that
makes it visible to predators, it is probably well
Males in many species o bird o paradise have adapted in other ways and is thereore a good
a complicated and eye-catching courtship dance mate to choose. Over the generations emales
that they use to try to attract emales. In some that selected males with showier plumage and
species the males all gather at a site called a lek more spectacular courtship dances have produced
and emales select a mate rom among the males ospring athered by males with greater overall
displaying. The coloured plumage and courtship tness. Natural selection has thereore caused
dances o birds help to avoid interspecic these traits to become exaggerated.
hybridization by allowing emales to determine i
a male belongs to their species, but this could be An example o a male bird o paradise can be seen
achieved in much more subtle ways than those in sub-topic 4.1 .
Changing learned and innate behaviour toK
Learned behaviour can spread through a population or be W ar sciniss
lost rom it more rapidly than innate behaviour. smims suspicius f
vidnc basd n amaur
Some patterns o behaviour are entirely innate, or example the bsrvains rar an
withdrawal refex, so are programmed into an animals genes. They can n numrica daa frm
happen immediately in an individual without any period o learning. cnrd xprimns?
However, they can only be modied by natural selection relatively
slowly because there must be variation in the alleles that aect the With respect to the
behaviour and a change in allele requencies in the population due to observations o the changes
one behaviour pattern increasing chances o survival and reproduction in the behaviour o blue tits
over the other patterns o behaviour. and milk bottles, an article
appeared in 1952 in the
Other patterns o behaviour are either partially or entirely learned journal Nature:
although these take longer to develop in an individual, they do not
involve changes in allele requency and can spread in a population Although no experimental
relatively rapidly as one individual learns rom another. C himpanzees analysis o the behaviour
show many examples o tool use that are learned, with considerable involved in the opening o
variation between groups o chimpanzees in the types o tool used. I one milk bottles has yet been
individual discovers a new use o an object as a tool, others can learn it made, urther observations
quickly. However, learned behaviour can also disappear rom a population in the feld enable the
rapidly. An example is blue tits eeding on cream rom milk bottles. discussion to be carried
urther.
553
A NEUROBIOLOGY AND BEHAVIOUR
Blue tits and cream
Feeding on cream from milk bottles in blue tits as an example of the development
and loss of learned behaviour.
B lue tits Cyanistes caeruleus were rst observed Much less milk is now delivered to doorsteps
pecking through the aluminium oil caps o because milk in supermarkets is cheaper. Also
milk bottles let outside houses, to drink the skimmed milk, without cream at the top, has
cream, in the 1 920s in Southampton, England. become popular with humans. This may explain
This behaviour was observed soon aterwards why blue tits have not recently been observed
1 50 kilometres away ar urther than blue tits pecking through bottle tops.
normally fy. Amateur birdwatchers ollowed the
rapid spread o the behaviour, in both blue tits
and great tits, across Europe to the Netherlands,
Sweden and Denmark.
German occupation o the Netherlands during the
Second World War stopped deliveries o milk or
eight years ve years longer than the maximum
lie o a blue tit. However, within months o the
resumption o deliveries, blue tits throughout the
Netherlands were pecking through the bottle tops.
The rapid spread o this behaviour pattern shows
that it must be due to learned rather than innate
b e h a v io u r.
Newspaper articles recently reported that blue tits Figure 6 Blue tit pecking through milk bottle cap
had stopped eeding on cream rom milk bottles.
554
QuestioNs
Questions c) Suggest two advantages to birds of [2]
keeping one eye open during sleep.
1 When birds are in danger of attack by predators,
they sometimes sleep with one eye open and 2 Alzheimers disease (AD) is characterized by
one eye closed. Neurobiologists investigated this increasing dementia (mental and emotional
behaviour pattern using mallard ducks (Anas deterioration) in affected persons.
platyrhynchos) . Video recordings were made of
groups of four sleeping birds, arranged in a row. Evidence from the post-mortem (after death)
The birds at the ends of the row were more analysis of the brains of affected patients has
vulnerable to predator attacks and kept one eye revealed two abnormalities. Affected persons show
open 1 50% more of the time than the two birds a change in the concentration of nerve growth
in the centre of the row. factor (NGF) in a region of the brain known as the
cortex. The brains of affected patients also have
Electroencephalograph (EEG) recordings were plaques. These are accumulations of insoluble
made to monitor the brain state of the birds material in and around cells.
at the ends of the rows. A region of the brain
which indicates whether the bird is asleep or A study was carried out to measure the post-
awake was monitored in each of the left and mortem NGF concentrations in two regions of
right cerebral hemispheres. EEG recordings the cortex, the temporal cortex and the frontal
were made when the birds were sleeping with cortex. Three groups of people were compared:
both eyes closed, when the birds had both eyes
open and also when they had one eye open. AD patients
These results are shown in the bar chart below,
as a percentage of the activity of the brain pre-AD patients with plaques but no
region when the birds were sleeping with both dementia
eyes closed.
a control group with no plaques and no
125 dementia.
left hemisphere
right hemisphere 140
100 120
activity of the brain region
(% of activity with both eyes closed) 100
% NGF of the control temporal cortex
80
75
60
0 both eyes both eyes left eye left eye 40
closed open closed right open right 20
eye open eye closed 0
Source: Rattenborg, et al. , Nature, 1999, 397, pages 397398 frontal temporal
a) State the effect of opening both eyes on AD patients pre-AD patients controls
activity in the region of the brain that was Source: R Hellweg et al., (1999) , International Journal ofDevelopment
Neuroscience, 16, (7/8) , pages 787794
being monitored. [1 ]
b) (i) Using the data in the bar chart, deduce
the effect on the two cerebral hemispheres a) Compare the data for the two regions
of the cortex.
of opening only the right eye. [2] [3]
(ii) Determine which hemisphere is more b) Calculate the increase in percentage NGF
awake when the right eye is open. [1 ]
in the frontal cortex of AD patients
(iii) Using the data in the bar chart, deduce compared to the control group. [1 ]
how the left and right eyes and left and
right hemispheres are connected. [1 ] c) Suggest what happens to the quantity of
NGF in the cortex as the disease
progresses. [2]
555
3AA N E U R O B I O LO G Y AN D B E H AVI O U R
3 Many animal species use long-range calls NN
to establish their use of space and their
relationships with members of their own and W E W E
other species. Most of the calls of the African 17:00h 2 4 6 8 10 18:00h 2 4 6 8 10
S avanna elephant ( Loxodonta africana) are
below the range of human hearing. The area S S
in which the elephants can detect the calls is N N
known as the calling area. O n any given day,
the calling area undergoes expansions and W E W E
contractions. The diagrams on the right show 19:00h 2 4 6 8 10 20:08h 2 4 6 8 10
the calling area (solid line) of elephants in
the Etosha National Park at different times of S S
the day. The position of the calling elephants N N
is the centre of the diagram. Circular rings
depict distance (in km) . The wind speed
(in m s-1) and direction are shown with an
arrow. If there is no arrow on the diagram it
shows there was no wind.
a) Identify the time of the day when the
calling area was greatest. [1 ]
b) Identify the wind speed at 08:00h. [1 ] EE
WW
2 4 6 8 10 2 4 6 8 10
c) Compare the calling area at 1 7:00h with
1 8:00h. [2]
d) Discuss the relationship between the 06:05h 08:00h
wind and the calling area.
[3] S S
Source: D Larom, et al. , Journal ofExperimental Biology
(1997) , 200, page 421431. Reprinted with the permission of
the Company of Biologists
556
B BIOTECHNOLOGY AND
C E LBLI OBIINOFLOO RGMY AT I CS
Introduction be used in the prevention and mitigation o
contamination rom industrial, agricultural and
Biotechnology is the use o organisms, especially municipal wastes. Biotechnology can also be
microorganisms to perorm industrial processes. used in the diagnosis and treatment o disease.
The organisms used may be genetically Bioinormatics is the use o computers to analyse
modifed to make them more suitable. Crops sequence data in biological research.
can be modifed to increase yields and to
obtain novel products. Biotechnology can
B.1 Microbiology: organisms in industry
Understanding Applications
Microorganisms are metabolically diverse. Deep-tank batch ermentation in the mass
production o penicillin.
Microorganisms are used in industry because
they are small and have a ast growth rate. Production o citric acid in a continuous
ermenter by Aspergillus niger and its use as a
Pathway engineering optimizes genetic and preservative and avouring.
regulatory processes within microorganisms.
Biogas is produced by bacteria and archaeans
Pathway engineering is used industrially to rom organic matter in ermenters.
produce metabolites o interest.
Skills
Fermenters allow large-scale production o
metabolites by microorganisms. Gram staining o Gram-positive and Gram-
negative bacteria.
Fermentation is carried out by batch or
continuous culture. Experiments showing zone o inhibition o
bacterial growth by bactericides in sterile
Microorganisms in ermenters become limited bacterial cultures.
by their own waste products.
Production o biogas in a small-scale ermenter.
Probes are used to monitor conditions within
ermenters. Nature of science
Conditions are maintained at optimal levels Serendipity has led to scientifc discoveries:
or the growth o the microorganisms being the discovery o penicillin by Alexander Fleming
cultured. could be viewed as a chance occurrence.
557
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
Figure 1 Penicillium mold growing on an Metabolic diversity
orange. The antibiotic penicillin is derived from
this microorganism Microorganisms are metabolically diverse.
Microorganisms occupy a number o niches in ecosystems. In order to
serve their ecological role, they require certain metabolic pathways that
correspond to their role.
Saprotrophs release nutrients trapped in detritus and make it available
to ecosystems. As saprotrophs, bacteria and ungi compete with one
another or ood sources. Many ungi release anti-bacterial antibiotics
into the environment in an eort to limit interspecifc competition.
Other microorganisms act as producers. Cyanobacteria (blue-green
algae) and protoctists such as algae and Euglena are photosynthetic. They
produce carbohydrates by fxing carbon dioxide in the Calvin cycle.
O ther microorgansims act as heterotrophs. Yeast such as Saccharomyces
cerevisiae carry out anaerobic respiration producing alcohol and carbon
dioxide by a pathway known as alcoholic ermentation.
The bacteria Rhizobium and Azotobacter can fx nitrogen and convert it
to a orm that living things can use. B acteria such as Nitrobacter and
Nitrosomonas can use inorganic chemicals as energy sources. They are
known as chemoautotrophs.
Humans have been able to take advantage o the metabolic pathways o
microorganisms in biotechnology applications.
Figure 2 Microalgae production for biofuels. The advantages of using microorganisms
Ponds being used to culture Chlorella vulgaris in biotechnology
microalgae as a source of biofuel. The carbon
dioxide is pumped into ponds (seen here) to Microorganisms are used in industry because they are
promote photosynthesis and therefore growth small and have a fast growth rate.
of the algae
Humans have been exploiting the metabolism o microorganisms
throughout history or example in the production o ood such as yogurt,
bread, wine and cheese.
More recently, industrial biotechnology has increased the number o
metabolic pathways exploited or drug and uel production as well as
additional applications involving genetically modifed microbes.
Industrial biotechnology takes advantage o the acts that
microorganisms are small and reproduce at a ast rate. They can be
grown on a range o nutrient substrates and can produce a range o
products. Conditions can be easily monitored in an industrial setting and
maintained at optimum levels.
Pathway engineering
Pathway engineering is used industrially to produce
metabolites of interest.
Traditionally either through selective breeding or genetic modifcation,
microorganisms used in biotechnology applications were selected
because they were the variants that provided the maximum yield o a
558
B .1 M i cro B i o lo g y: o rg an i s M s i n i n d u s try
desired metabolite. What this didnt take into account was the possibility
that there were points in the metabolic pathway that constrained yields
to the point where actual yields were much lower than theoretical yields.
What distinguishes pathway engineering rom traditional methods is
the use o detailed knowledge and analysis o the cellular system o
metabolic reactions. This allows scientists to direct changes at multiple
points to improve yields o metabolites o interest. This can include
extending the range o substrates, elimination o by-products that slow
the process down and extension o the range o products.
Pathway engineering uses knowledge of Figure 3 Coloured scanning electron
metabolic pathways to increase yields micrograph (SEM) of naturally occuring
yeast cells (red) on the skin of a grape. In
Pathway engineering optimizes genetic and regulatory the processing of the grapes to make wine,
processes within microorganisms. the presence of the yeast is essential for the
fermentation of the grapes that is part of the
Pathway engineering is a technique that analyses the metabolic pathway wine making process
o a particular microorganism to determine the bottleneck points o
the pathway that constrain the production o the desired compound. 559
Researchers can then address the constraint using genetic modifcation.
For example, the yeast Saccharomyces cerevisiae occurs naturally on the
skin o grapes. The ermentation o grapes is carried out by S. cerevisiae
with the desired end product being ethanol. Maintaining the correct
pH is important in wine production. Malate is a metabolite that appears
during wine making. Its degradation is essential or the deacidifcation
o grapes. However, malate permease, a membrane protein necessary or
the transport o malate into cells is not present in S. cerevisiae. Further,
while S. cerevisiae has an enzyme that can degrade malate, it was ound
to be relatively inefcient.
The gene or MAE2, a highly efcient malate degrading enzyme rom
Lactococcus lactis was inserted into S. cerevisiae along with the gene or
malate permease rom the yeast Schizosaccharomyces pombe. The ability o
transgenic S. cerevisiae to undertake more efcient malate degradation
was successully achieved.
Fermenters in industry
Fermenters allow large-scale production of metabolites
by microorganisms.
Technically, ermentation reers to the anaerobic generation o ATP
rom glucose that generates characteristic end products such as alcohol
and lactic acid. With respect to biotechnology, microbiologists have a
broad interpretation o the term ermentation; i.e., the word reers to
the processes involved in the large-scale culture o microorganisms to
produce metabolites o interest.
A ermenter is oten a large stainless steel vessel flled with sterile
nutrient medium. The medium is inoculated with the desired
microorganism. An impeller is a rotating set o paddles that mixes the
medium preventing sedimentation. Gas is bubbled through i the desired
metabolic process is aerobic. A pressure gauge detects gas build-up and
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
toK allows waste gases to escape. Conditions within the vessel are monitored
by probes. Because heat can build up as a waste product o metabolism,
t wha exen is scienifc a cooling jacket surrounds the reaction vessel with cooling water fowing
develpmen dependen n through it. Once the medium is used up, new medium can be added.
lucky accidens? Product removal may also occur leading out o the vessel.
In 1897, Hans and Eduard
Buchner were investigating antifoam motor a cid /ba s e
yeast extracts as a source pressure guage
of medicine. They ground up steam
yeast cells with silica and nutrient or inoculant
sand and used a hydraulic
press to create a yeast sterile nutrient medium ltered waste gases
extract. They applied high cold-water outlet
concentrations of sugar to impeller
serve as a preservative. oxygen concentration probe pH probe
What surprised them was temperature probe
this cell-free system began
to ferment the sugar. Eduard cooling jacket
Buchner received the Nobel
Prize for his discovery of cell- cold-water inlet sparger
less fermentation. This had steam compressed air
actually been discovered
in 1878 by Wilhelm Kuhne, Figure 4 A fermenter harvest pipe
but he had not been
successfully able to isolate There are two approaches to industrial
the chemical element in fermentation
the way that the Buchners
did. Kuhne did provide the Fermentation is carried out by batch or continuous culture.
name for the element that
was causing the cell-less Mass culture o microorganisms is carried out in two ways in industry.
ferementation contained
in the yeast. He created Batch culture is used or producing secondary metabolites; i.e., those which
the term enzyme from the are not essential or the growth o the culture. In this case, inoculation o
Greek words en (in) and the medium is ollowed by the culture passing through all o the stages o
zume (yeast) . the sigmoid growth curve. To begin the process, a xed volume o medium
is added to the closed ermentation vat. Ater inoculation, no urther
560 nutrients or microorganisms are added during the incubation period. The
products are extracted only when they reach a high enough concentration.
In continuous culture, nutrients are added and products harvested at a
constant rate. Conditions are monitored closely and eorts are made to keep
conditions at a constant level so the process can continue over a long period.
Factors limiting industrial fermentation
Microorganisms in fermenters become limited by their
own waste products.
A number o abiotic actors set limits to the activity o microorganisms
in ermentation tanks. These can be due to the consumption o raw
materials by the microorganism or by the production o waste products
due to their activities.
B .1 M i cro B i o lo g y: o rg an i s M s i n i n d u s try
C arbon dioxide production can lower the pH aecting enzyme activity.
Gas production can lead to pressure build-up possibly aecting
reaction rates.
Alcoholic ermentation can yield levels o alcohol which have an
osmotic eect on cells.
Oxygen levels can be depleted due to cellular respiration.
Heat as a waste product o metabolism can raise the temperature o
the reaction vessel.
Probes monitor conditions within fermenters
Probes are used to monitor conditions within fermenters.
In gure 5, oxygen concentration, pH, volume, oam levels and
temperature probes are shown. These are the most commonly monitored
variables in ermentation tanks. C omputer-based probes gather data on
these conditions. In batch ermentation, they can provide an indication
o the stage o the production process. In continuous ermentation, they
can signal to a technician actions to be taken to keep conditions within
the avourable range.
Maintaining optimum conditions within Figure 5 A system of probes is connected to
fermenters the fermenter to monitor conditions within
the vessel
Conditions are maintained at optimal levels for the growth
of the microorganisms being cultured.
Conditions are more likely to be monitored and kept at optimal levels in
continuous culture. Such conditions include water content, temperature,
pH, macro- and micro-nutrient levels, levels o waste products, cell
density, dissolved oxygen content, dissolved carbon dioxide content,
culture volume and culture mixing. The optimum level o each variable
depends on the species.
The level o a variable is oten infuenced by a number o variables and
is thereore important to monitor constantly. C onsider the example o
oxygen. S pecies dier in their tolerance o low oxygen. Penicillium is
less tolerant o low oxygen than Saccharomyces. When concentrations
o dissolved oxygen go below a critical value, then it becomes limiting.
Dissolved oxygen is aected by the temperature and the nutrients being
oxidized by the organism. Adding oxygen by aeration to a culture is not
a simple matter as it generates oam which can limit production. Anti-
oaming agents are oten added to the reaction vessel.
Deep-tank fermentation
Deep-tank batch fermentation in the mass production of penicillin.
In the early 2 0th century, eorts were concerted However, this did not produce signicant enough
to nd ways to mass produce penicillin. Initial yields to meet the demands or treatment o the
experiments showed that Penicillium notatum grew casualties o World War II. Large-scale production
best in shallow pans due to the need or aeration. was acilitated by deep-tank ermentation. This
561
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
employed both a source o oxygen bubbled in to In the case o penicillin, optimum conditions are
the tank and paddles to distribute the oxygen. The about 24 C, slightly basic pH and a good oxygen
nutrient source or the Penicillium is corn steep supply. The product typically starts being ormed
liquor. This is the liquid produced by warming a about 30 hours ater the start o the batch culture
vat o corn in water near 50 C or two days. as nutrient concentrations begin to decline
and continues or about six days ater which
Antibiotics are secondary metabolites in the sense the ermenter has to be drained and the liquid
that they are produced at a certain point in the ltered. Using solvents, a crystalline precipitate is
lie cycle o the microbe under certain conditions. generated rom the ltered liquid.
Industrial production of citric acid
Production o citric acid in a continuous ermenter by Aspergillus niger and its use
as a preservative and favouring.
Citric acid is an important ood additive, both as produced in the Krebs cycle and so is reerred to
a favour enhancer and a preservative. Industrial as a primary metabolite. I the culture medium is
production o citric acid relies on the ungus under-supplied with certain minerals such as iron,
Aspergillus niger. While the greatest raction o citric acid builds up in the reaction vessel.
industrially produced citric acid is produced by
batch ermentation, continuous ermentation has Ater the contents o the ermentation vessel are
also been attempted. The optimal conditions or ltered out, calcium hydroxide is added to the
citric acid production are high dissolved oxygen ltrate and solid calcium citrate precipitates out o
concentration, high sugar concentration, an acidic solution. It can then be urther treated chemically
pH and a temperature o about 30 C. Citric acid is to yield citric acid.
Gram staining
Gram staining o Gram-positive and Gram-negative bacteria.
A traditional test used to classiy bacteria is whether peptidoglycan (a polymer consisting o amino acids
they are Gram-negative or Gram-positive, based on and sugars) . The greatest raction o the Gram-
how they react to Gram-staining. The cell wall o positive cell wall is composed o peptidoglycan.
Gram-positive bacteria consists o a thick layer o
acidic polysaccharides lipopolysaccharide-
thick peptidoglycan layer rich outer envelope
plasma membrane
thin peptidoglycan layer
plasma membrane
(a) gram-positive: thick cell wall, no outer envelope (b) gram-negative: thinner cell wall, with outer envelope
Figure 6
562
B .1 M i cro B i o lo g y: o rg an i s M s i n i n d u s try
The cell wall o Gram-negative bacteria is much crystal violet stain with it. In contrast, the crystal
thinner only about 20% peptidoglycan (see violet binds to the multiple layers within the thick
fgure 6) . Crystal violet binds to the outer membrane peptidoglycan layer which is not washed away by
in Gram-negative bacteria and when alcohol is the alcohol and thus the colour persists.
added, it washes away the outer membrane and the
av: gram positive gram negative
gm- pee xation
1 Prepare smears of Bacillus cereus, Streptococcus
crystal violet
fecalis, Escherichia coli and Micrococcus luteus. Fix
these preparations by heating over a bunsen burner. iodine treatment
2 Stain with crystal violet for about 30 seconds. decolorization
3 Rinse with water, then cover with Grams iodine. Allow counter stain
stain to act for about 30 s. sa fra n i n
4 Rinse with water, then decolorize with 95% alcohol
for 1020 s.
5 Rinse with water, then counterstain with safranin
for 2030 s.
6 Rinse with water and blot dry. Gram-negative bacteria
will be pink. Gram-positive bacteria will be blue or violet.
7 Depending on local restrictions, you might choose to
examine prepared slides of Gram-negative and
Gram-positive bacteria.
Biogas production
Biogas is produced by bacteria and archaeans from organic matter in fermenters.
Biogas reers to the combustible gas produced CH3COOH CH4 + CO2
rom the anaerobic breakdown o organic matter
such as manure, waste plant matter rom crops (splitting ethanoic acid to orm methane and
and household organic waste. Depending on the carbon dioxide)
construction o the ermenter, biogas is mostly
methane with some carbon dioxide, though other IN OUT
gases may be present.
sewage from people methane for
Three dierent communities o anaerobic manure from animals cooking, heating
microbes are required. The frst group convert the farm waste or refrigeration
raw organic waste into a mixture o organic acids, garden waste
alcohol, hydrogen and carbon dioxide. The second
group use the organic acids and alcohol rom OUT
the frst stage to produce acetate, carbon dioxide
and hydrogen. These frst two communities Slurry, which can be
are Eubacteria. The last group are Archaea used as a fertilizer
called methanogens. The methanogens produce
methane by one o the ollowing two reactions:
CO2 + 4H2 CH4 + 2H2O Figure 7 Methane generator. Conditions inside
(reduction o carbon dioxide to methane) must be anaerobic
563
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
Producing biogas in the classroom
Production o biogas in a small-scale ermenter.
Figure 8 shows an example o a set-up o a biogas o organic waste and water could be compared in
generator. Mylar balloons are the ones that are terms o rate o biogas production.
commonly flled with helium as party balloons.
The eedstock bottle should be plastic rather than rubber tube the end ofthis tube
glass due to the risk o explosion. The tube clamps tube clamps must be ABOVE the
can be used to prevent gas leakage when the mylar balloon level of the feedstock
balloon is to be disconnected rom the set-up. The
balloon should be sealed to the glass tube with feed stock
insulating tape.
glass connector tubes
The rate o biogas generated by dierent
eedstocks could be compared. Relative quantities seal mylar balloon to glass tube with insulating tape
Figure 8
Serendipity and the discovery o penicillin
Serendipity has led to scientifc discoveries: the discovery o penicillin
by Alexander Fleming could be viewed as a chance occurrence.
Serendipity is defned as a lucky accident or o his bacterial plates was contaminated with
the situation where something good or useul ungus and the zone around the ungus on the
is revealed when it was not being specifcally plate appeared to have no bacteria while urther
searched or. However, it is only useul i the away rom the ungus, bacteria grew on the
observer recognizes its value. plate. Fleming was wise enough to connect the
unexpected observation to his earlier studies o
Alexander Fleming was a Scottish medical anti-bacterial agents.
doctor and scientist who spent the early part o
his career searching or anti-bacterial agents. He proceeded to grow the mold in pure culture
In 1 928, he was investigating the properties o and then test it on a number o pathogenic
the bacterium Staphylococcus. Ater returning bacteria and discovered that it had an antibiotic
rom an extended holiday, he noticed that one eect on several species.
Zones o inhibition as a measure o bactericide efectiveness
Experiments showing zone o inhibition o bacterial growth by bactericides
in sterile bacterial cultures.
Bacteria are oten grown on nutrient media in
glass or plastic plates called Petri dishes. The
plates are incubated under laboratory conditions.
Lids are kept on the plates in order to prevent
contamination. Individual bacteria divide and
orm colonies, but i the entire nutrient surace
is exposed to the bacterium, then a lawn o
bacteria is grown.
What Fleming observed is known as a zone o Figure 9
inhibition; that is, a region on a bacterial lawn
564
B. 2 Bi otech n o lo g y i n agri cu ltu re
where an anti-bacterial eect prevents the growth individuals but is a major cause o inections
o bacteria. The consequence is an oten circular- acquired by people in hospitals.
shaped disc region. The diameter o the zone
o inhibition is a measure o the strength o the This technique can be modifed by students to
anti-bacterial agent. In fgure 9 a plate inoculated investigate the eectiveness o various anti-
with Pseudomonas aeruginosa bacteria had various bacterial agents. Absorbent flter paper can be cut
types o antibiotic discs placed on the surace to into disc shapes by a hole puncher. The discs can be
determine which is the most eective. This is soaked in disinectants, or example, and placed on
a species o bacteria that rarely inects healthy to a plate that has been inoculated with bacteria.
B.2 B
Understanding Applications
Transgenic organisms produce proteins that were Use o tumour-inducing (Ti) plasmid o
not previously part o their species proteome. Agrobacterium tumefaciens to introduce
glyphosate resistance into soybean crops.
Genetic modifcation can be used to overcome
environmental resistance to increase crop yields. Genetic modifcation o tobacco mosaic virus to
allow bulk production o Hepatitis B vaccine in
Genetically modifed crop plants can be used to tobacco plants.
produce novel products.
Production o Amora potato (Solanum
Bioinormatics plays a role in identiying tuberosum) or paper and adhesive industries.
target genes.
Skills
The target gene is linked to other sequences
that control its expression. Evaluation o data on the environmental impact
o glyphosate-tolerant soybeans.
An open reading rame is a signifcant length o
DNA rom a start codon to a stop codon. Identifcation o an open reading rame (ORF) .
Marker genes are used to indicate successul Nature of science
u p ta ke .
Assessing risks and benefts associated with
Recombinant DNA must be inserted into the scientifc research: scientists need to evaluate
plant cell and taken up by its chromosome or the potential o herbicide resistant genes
chloroplast DNA. escaping into the wild population.
Recombinant DNA can be introduced into whole
plants, lea discs or protoplasts.
Recombinant DNA can be introduced by direct
physical and chemical methods or indirectly
by vectors.
565
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
Figure 1 Transgenic organisms
Transgenic organisms produce proteins that were not
previously part o their species proteome.
The complete set o proteins that a cell or organism can make is reerred
to as its proteome. Proteins are key components o a cells structure
and carry out most cellular unctions. Sometimes genetic engineers
seek to extend the proteome o an organism or the purposes o a
biotechnological application. I the addition to the proteome is due to
the addition o a gene rom a dierent organism, then the modied
organism is said to be transgenic.
Figure 1 shows glo-sh, the rst genetically modied organism to be
sold as a pet. These transgenic sh have had the gene or the production
o green fuorescent protein introduced into their genome. The original
organism that was the source o the gene was Aequorea victoria, a j ellysh.
The protein SRY is a transcription actor which triggers the expression
o genes that lead to the development o male characteristics. Figure 2
shows a transgenic emale mouse (on the right) that has been genetically
modied to express the protein SRY within its proteome. It has caused
the emale mouse to develop the same genetalia as the male on the let.
Figure 2 Genetically modifed crop plants
Genetically modifed crop plants can be used to produce
novel products.
A novel product reers to the presence o a protein or phenotype that
was not previously ound in the species.
The production o golden rice involved the introduction into rice o
three genes, two rom daodil plants and one rom a bacterium, so that
the orange pigment -carotene is produced in the rice grains. -carotene
is a precursor to vitamin A. The development o golden rice was
intended as a solution to the problem o vitamin A deciency, which is a
signicant cause o blindness among children globally.
Corn has been genetically modied to produce the CRY toxin due to
the insertion o a gene rom Bacillus thuringiensis. As a consequence, the
plant becomes unpalatable to the European corn borer, an insect pest
that signicantly reduces crop yields.
Overcoming environmental resistance in crops
Genetic modifcation can be used to overcome
environmental resistance to increase crop yields.
Limiting actors aecting crop plant growth can be biological or
no n- b io lo gical.
Biotic actors include competition rom weed species, predation by
insects and inection by pathogens.
566
B. 2 Bi otech n o lo g y i n agri cu ltu re
Resistance to the herbicide glyphosate has been introduced to crop plants
such as soybeans as part o a strategy or reducing competition with weeds.
The introduction o genes or the production o Bt toxin into corn is part
o a strategy or reducing predation by insects such as the western corn
rootworm. Non-transgenic roots will suer considerable damage rom
pests, but transgenic roots suer little damage as they have resistance to
the rootworm due to the expression o the Bt toxin.
In Hawaii, researchers genetically modifed papaya plants to be resistant
to papaya ringspot virus by leading the plant to express the gene or the
virus coat triggering a protective response to the virus.
Abiotic actors that limit crop growth include such actors as drought,
rost, low soil nitrogen and high soil salinity.
D roughtGard maize contains the gene or cold shock protein B ( cspB)
rom the bacterium Bacillis subtilis that enables it to retain water during
drought conditions.
A gene rom Thale cress ( Arabidopsis) , AtNHXI, codes or the production
o a membrane protein that captures excess sodium into plant vacuoles.
Peanut plants have been genetically modifed to express this gene
allowing them to grow in saline soils that would otherwise limit
crop output.
Components of the gene construct Figure 3 Transgenic sheep, awaiting milking.
The sheep are ospring o ewes which have
The target gene is linked to other sequences that control a human gene responsible or the production
its expression. o the protein alpha-1-antitrypsin (A1AT)
incorporated into their DNA. A1AT is produced
To carry out genetic modifcation, more than the gene must be inserted. in mammary cells, and secreted in the sheep's
Additional sequences are necessary to control the expression o the gene. milk. The A1AT can then be isolated and used
to treat hereditary A1AT defciency in humans,
Most commonly, sequences such as a eukaryotic promoter must be which leads to the lung disease emphysema
added upstream in the construct and a eukaryotic terminator sequence
must be included downstream in the construct. The construct also oten
contains a second gene called a recognition sequence which allows
engineers to confrm that the construct has been taken up by the host
DNA and is being expressed.
In some cases, specifc additional sequences have to be added. Consider
the example o genetically modiying sheep to express human proteins
such as alpha-1 -antitrypsin in the sheeps milk. In this case, a specifc
promoter sequence that will ensure that the gene is expressed in
milk is necessary in creating the gene construct. In addition, a signal
sequence has to be added to ensure that the protein is produced by
ribosomes on the endoplasmic reticulum rather than by ribosomes that
are ree in the cytoplasm. This is to ensure that the alpha-1 -antitrypsin
protein is secreted by the mammary cells rather than released
i n t r a c e l l u l a r l y.
567
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
Figure 4 Marker genes
Marker genes are used to indicate successful uptake.
In addition to the target gene, an additional gene is oten added to provide
some way to indicate that uptake o the target gene has occured. Some
markers are based on articial selection. In this case, the gene is called a
selectable marker. The marker gene oten coners antibiotic resistance. Those
bacteria that have taken up the marker gene and the target gene construct
will survive exposure to antibiotic. These can then be cultured separately.
The gene or the production o green fuorescent protein (GFP) is also
used as a marker. Figure 4 shows mosquitos that have been genetically
modied to resist being hosts or the malarial parasite. The donor gene
has been linked to the gene or GFP so that the transgenic mosquitos can
be detected under a microscope.
Recombinant DNA
Recombinant DNA must be inserted into the plant cell and
taken up by its chromosome or chloroplast DNA.
Recombinant DNA is a molecule that has been manipulated so that it
contains sequences rom two or more sources.
In order to create a transgenic organism, the recombinant DNA must be
taken up by the host cell.
In order or the gene to be expressed, it has to be taken up into a
chromosome. In plant cells, it can also be taken up by a chloroplast.
This process o uptake and expression o the donor DNA is called
transormation. The new genes can be inserted into the DNA o the
chloroplasts. The major advantage o inserting into chloroplasts is
that the chloroplast DNA is not transmitted through pollen which
prevents gene fow rom the genetically modied plant to other plants.
Transormation usually requires the use o a vector.
Diferent targets or genetic transormation
Recombinant DNA can be introduced into whole plants,
leaf discs or protoplasts.
Once the transgene has been introduced into the host cell, the production
o a whole plant rom the transormed cell has to be perormed.
Protoplasts are plant cells that have had their cell walls removed.
Transormation by Agrobacterium was initially attempted on protoplasts.
While this was somewhat successul, the diculty o obtaining sucient
high quality protoplasts combined with the diculty o growing whole
plants rom protoplasts led to the search or other methods.
The lea disc methods involves incubating lea cut-outs with
Agrobacterium containing a plasmid with the target gene along with an
antibiotic resistance gene. The lea discs are then transerred to a plate
containing two dierent antibiotics which ensures that only transormed
cells will grow. The transormed cells are then cultured and treated in
such a way so that roots and shoots develop rom the discs.
568
B. 2 Bi otech n o lo g y i n agri cu ltu re
Diferent methods o genetic transormation
Recombinant DNA can be introduced by direct physical
and chemical methods or indirectly by vectors.
Genes can be introduced into plants in a number o dierent ways
including microinjection, electroporation, virus inection, ballistic
incorporation and incubation with Agrobacterium tumefaciens.
Incubating host cells at cold temperatures in calcium chloride solution
and then heat shocking the solution is a chemical method that was one
o the original methods or transorming cells.
Electroporation is a physical method that involves applying an external
electric feld that leads to the ormation o temporary pores in the cell
membrane allowing recombinant DNA to get into a cell.
Microinjection is another physical method o introducing genes. A
pipette is used to aspirate and hold a cell in a fxed position while a
needle is used to inject genes o interest.
In biolistics, metal particles coated with the gene o interest are fred at
an entire plant.
A vector is a virus, a plasmid or some other biological agent that
transers genetic material rom one cell to another. In the next section
the use o the Ti plasmid vector is explained. On page 570 the use o a
virus as a vector is explained.
The use o Ti plasmid as a vector
Use of tumour-inducing (Ti) plasmid of Agrobacterium tumefaciens to introduce
glyphosate resistance into soybean crops.
One way to introduce transgenes into plants is to gene. The construct is then re-inserted into an
use Agrobacterium tumefaciens. This is a species o A. tumefaciens bacterium. Plant cells are then
bacteria that has a plasmid, called the Ti plasmid, exposed to the transgenic bacterium and cultured
that causes tumours in the plants it inects. on a plate containing antibiotic. The only plant
cells that grow are those that have taken up the
The glyphosate resistance gene is inserted into plasmid. The others are killed by antibiotic.
the Ti plasmid along with an antibiotic resistance
glyphosate
resistance gene bacterial cell plant cell gene transfer
plasmid DNA
bacterial
antibiotic resistance gene suspension dead cell callus
1 Cut leaf 2 Expose leaf to bacteria antibiotic medium 4 Allow callus to 5 The plants
carrying an antigen sprout shoots and are transferred
Figure 5 gene and an antibiotic 3 Expose leaf to an roots to soil where they
resistance gene. Allow antibiotic to kill cells can develop into fully
bacteria to deliver the that lack the new genes. dierentiated adult plants
genes into leaf cells Wait for surviving that are glyphosate resistant
(gene-altered) cells to
multiply and form a 569
clump (callus)
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
Edible viruses Capsid gene Hepatitis B gene coding
antibodies for antigen that will
Genetic modifcation o tobacco mosaic stimulate an immune
virus to allow bulk production o response
Hepatitis B vaccine in tobacco plants. +
Capsid gene for tobacco
Vaccination programmes are oten impacted mosaic virus (TMV)
by lack o access to remote areas as well as the
challenge o rerigerating vaccine preparations. Fusion of two genes and
One initiative has been to develop edible vaccines incorporation into virus
by incorporating antigens into plant matter. O ne
attempt involved genetically modiying tobacco Hepatitis B gene
mosaic virus with antigens rom the Hepatitis B
virus and then inecting tobacco plants. Infect plant Plant expresses
the antigen
Feed to the animal whose
immune system responds by
creating antibodies to the
Hepatitis B virus
Figure 6
Potatoes modifed to produce starch containing only amylopectin
Production oAmora potato (Solanum tuberosum) or paper and adhesive industries.
Potatoes are used in industry as a source o o starch polymers (see fgure 7) . 80% o
starch. Starch can be used or a number o potato starch consists o the branched chain
purposes including use as an adhesive. Normally, amylopectin and 20% is the straight chain orm,
potato starch consists o two dierent types amylose.
HO OH
OO
HO HO O O OH OH O OH
O O
HO HO O O HO HO O HO O
HO
HO HO O HO O
amylose O
HO
HO O O
OH
HO HO O O
HO O
amylopectin HO
Figure 7
570
B. 2 Bi otech n o lo g y i n agri cu ltu re
When a starch mixture is heated and then cooled, rather than being translated to orm protein
it tends to orm a gel which is undesirable or
some applications such as paper manuacturing (gure 8).
and adhesive production. To prevent this,
conventional methods use chemical treatment to antisense strand digestion
remove the amylose.
RNase
The company BASF produced a genetically
modied potato where one o the genes involved transcription translation
in the production o amylose was deactivated.
The gene product was granule bound starch DNA duplex
synthase. The method used was antisense formation
technology. This involves inserting a version o mRNA
the gene that is inverted such that it produces the
antisense mRNA. The result would be that the Figure 8
normal sense strand would be produced as well as
the antisense strand. The two would bind and the
double stranded mRNA molecule gets degraded
Assessing risks of transgenes entering wild populations
Assessing risks and benefts associated with scientifc research: scientists need to
evaluate the potential o herbicide resistant genes escaping into the wild population.
Gene fow is the movement o genes or genetic dicult. I the transgene is or insect resistance,
material rom one population to another. In plant then ecological balance could be disrupted.
populations, it can occur through the transer o
pollen between related species. Assessing the risk requires estimating how
requently gene fow occurs, determining whether
Herbicide resistant genetically modied (GM) the transgene becomes expressed and determining
crops are the most common type o genetically the changes to the phenotype o the plant.
modied crop grown. One strategy or reducing risk is to incorporate
mitigator genes with the transgene which is
The potential fow o transgenes rom the GM designed to reduce the success o any hybrid
crop to non-GM crops and rom the GM crop to plants that might be accidentally created. Another
wild weed populations is an economic concern. strategy is to transorm chloroplasts rather than
I the transgene becomes expressed in the wild nuclear DNA as the chloroplast DNA is not
population, then controlling the eect o the weed expressed in pollen.
population within a crop area would become
Evaluating the environmental impact of a GM crop
Evaluation o data on the environmental impact o glyphosate-tolerant soybeans.
Weeds reduce crop yields by competing with crop modied to be glyphosate resistant allowing
plants or space, sunlight, water and nutrients. armers to use a single broad-spectrum herbicide.
Glyphosate is a chemical that kills a very broad
spectrum o plants. Soybeans as well as a There are two potential environmental aspects
number o other crop species have been genetically to consider: the environmental risks o the
genetic modication o a crop plant and the
571
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
environmental risks o the widespread use o the ossil uel use required or tillage and reduced
glyphosate as an herbicide that is encouraged by the need or inputs required to supplement soil
the prevalence o the GM crop. ertility. Figure 9 shows the growth in the area
cultivated with GM soybeans in Argentina with a
There has been broad academic consensus that there corresponding growth in no-till agriculture.
has been at least some environmental beneft o
genetically modifed glyphosphate-tolerant crops Glyphosate resistance (GR) is under intense
in replacing previous widespread systems o weed selection pressure given the widespread use o the
reduction. The beneft to this modifcation is that crop crop and the reduced use o other herbicides. The
weeds can be controlled by herbicide without the risk environmental consequences o resistant weeds
o reduced crop yields because the crop is resistant to will include reduced crop yields or the same
the herbicide. Further the level o herbicide required inputs and the need to increase the use o tillage
to be applied is lower (table 1 ) than beore the GM and alternative herbicide ormulations.
crop was introduced. While the data is disputed,
many researchers claim that glyphosate is nearly the A review conducted in 2002 by the European
least toxic pesticide used in agriculture. Union reached the conclusion that there was
little data to support claims o health impacts o
georaphic reion % chane in herbicide use in glyphosate on humans. Some studies suggest
that other components o the herbicide mixture
comparison to non-gM crops in 1997 used in combination with glyphosate did
have environmental impacts. The Australian
Heartland -23% government has banned the use o some
ormulations o glyphosate near water.
Northern Crescent -15%
Mississippi Portland -11%
Southern Seaboard -51%
Table 1 Percentage reduction in the amount o herbicide GM soybeans (million ha)18GM soybean 25
applied in genetically modifed crops over traditional crops in no-till farming (million ha)16no-till farming20
various regions o the US 14 15
12 1998 2000 2002 10
Tillage is the practice o turning over the soil and 10 5
has been commonly practised as a component 0
o weed management strategies. The loss o 8
top soil and erosion is one o the consequences 6 2004
o tillage. Glyphosate and glyphosate-resistant 4
crops have enabled signifcantly less tillage and 2
thereore preserved soil ertility. This has reduced 0
1996
Figure 9
Open reading frames
An open reading rame is a signifcant length o DNA rom
a start codon to a stop codon.
When the DNA o an organism has been sequenced, researchers will
then look or the location o genes. The starting point or this search is to
look or open reading rames.
The search or open reading rames (ORF) depends on the ollowing concepts:
There are 64 triplets o bases that are called codons.
61 codons are used to code or an amino acid.
There are 3 three stop codons ( TAA, TAG and TGA) that signal the
end o an open reading rame.
572
B. 2 Bi otech n o lo g y i n agri cu ltu re
There is one start codon ( ATG) that signals the start o an open
reading rame and also codes or an amino acid.
Open reading rames in DNA are identifed by searching or base
sequences long enough to code or the amino acids in a polypeptide
between a start codon and one o the three stop codons. In other words,
they look or sequences where stop codons are absent. Researchers
usually look or a base sequence long enough to code or one hundred
amino acids. The average size o an O RF in E. coli is 3 1 7 amino acids long.
Identifying open reading frames
Identifcation o an open reading rame (ORF) .
A short base sequence is shown below. 2 Researchers need to distinguish between open
AATTC ATGTTC GTC AATC AGC AC C TTTGTGGTTC reading rames that code or polypeptides and
TC AC C TC GTTGAAGC TTTGTAC C TTGTTTGC GGT random base sequences in the genome that
GAAC GTGGTTTC TTC TAC AC TC C TAAGAC TTAA by chance have start codons ollowed by an
TAGC C TGGTG extended sequence without a stop codon.
1 Find the frst start codon and the frst stop a) Calculate the percentage chance o
codon ater it in the sequence. fnding a start codon in a piece o
DNA with a random sequence o ten
a) State how many bases there are beore base pairs. [2]
the start codon. [1 ] b) I the start codon is ound in a random
b) State how many codons there are in base sequence, calculate the percentage
the open reading rame that you have
ound. chance that the next triplet o bases
[1 ] codes or an amino acid. [1 ]
c) Calculate how many amino acids are c) Calculate the percentage chance [2]
encoded in this open reading rame. that the next 1 00 triplets all code or
Show how you worked out your answer. [3] amino acids.
d-bs qss: Determining an open reading frame
Once the sequence o bases in a piece o DNA 2 Determine the raction o codons that are stop
has been determined, a researcher may want codons in the genetic code. [2]
to locate a gene. To do this, computers search 3 In table 2, the codons could start with the
through the sequences looking or open reading
rames. An open reading rame is one that is frst, second or third base. These correspond
uninterrupted by stop sequences and could
thereore code or the production o a protein. to three dierent reading rames (RF1 , RF2
The stop codons are UGA, UAA and UAG.
or RF3) . Determine which o the reading
rames, 1 , 2 or 3, might be an open
reading rame. [2]
1 State the number o codons in the
genetic code. [1 ]
dna 3' A T T A A C T A T A A A G A C T A C A G A G A G G G C T A G T A C
mrna 5'
rF1 U A A U U G A U A U U U C U G A U G U C U C U C C C G A U C A U G
rF2 A A U U G A U A U U U C U G A U G U C U C U C C C G A U C A U G
rF3 A U U G A U A U U U C U G A U G U C U C U C C C G A U C A U G
Table 2
573
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
aciviy organisms GI number. It is listed in the title. (GI number
#110832861) . View the genome.
Alcanivorax borkumensis is a rod-shaped bacterium
that utilizes oil as an energy source. It is relatively Go to the open reading rame nder (http://www.ncbi.nlm.
uncommon but quickly dominates the marine microbial nih.gov/projects/gor/) . Enter the GI number and speciy
ecosystem ater an oil spill. Scientists sequenced the the range o bases that you are going to search.
genome o this bacterium in an efort to identiy the
genetic aspects o its oil digesting ability. The entire Perhaps as a class, the genome can be divided up into
genome can be accessed rom the database GenBank. 2000 bp pieces. Share inormation with one another
about the open-reading rames identied.
Visit GenBank and search by genome to locate the
genome o this organism. Click on FASTA to identiy the
toK Identifying target genes
Wh knwledge issues re Bioinormatics plays a role in identiying target genes.
creed by he rpid grwh
in he mun f vilble Bioinormatics is the use o computers to investigate biological
d nd infrmin? phenomenon. Open reading rames are identifed by subjecting genomic
inormation held in a database to searches to fnd extended sequences
The technology o without stop codons.
DNA sequencing and
bioinormatics has evolved Once an open reading rame is identifed, a BLAST search can be
at a rapid pace. In 2009, conducted. The acronym reers to Basic Local Alignment Search Tool. A
the biggest problem or BLASTn search would search through databases to determine i an open
researchers was developing reading rame with a similar nucleotide sequence existed in another
solutions to improve the species. A BLASTx search would search a protein database based on the
sequencing o DNA. Time and translated sequence o the open reading rame.
cost limited the production o
DNA sequence inormation. Alternatively, i a researcher has ound a protein and wants to determine
By 2013, researchers can the location o a gene, they can conduct a tBLASTn search using a
sequence a whole human computer search o multiple genomes using the translated sequence to
genome within a single search or potential genes that could have been transcribed to produce
day. The challenge has now the protein. All three methods play a role in identiying target genes.
shited rom sequencing DNA
to managing and analysing
the extraordinary volume
o sequence data that is
being produced. It has been
estimated that ve months
o analysis are needed or
every month's worth o data
generated.
574
B.3 environMental protection
B.3 em
Understanding Applications
Responses to pollution incidents can involve Degradation o benzene by halophilic bacteria
bioremediation combined with physical and such as Marinobacter.
chemical procedures.
Degradation o oil by Pseudomonas.
Microorganisms are used in bioremediation. Conversion by Pseudomonas o methyl
Some pollutants are metabolized by mercury into elemental mercury.
microorganisms. Use o bioflms in trickle flter beds or sewage
Cooperative aggregates o microorganisms can treatment.
orm bioflms.
Skills
Bioflms possess emergent properties.
Evaluation o data or media reports on
Microorganisms growing in a bioflm are highly environmental problems caused by bioflms.
resistant to antimicrobial agents.
Nature of science
Microorganisms in bioflms cooperate through
quorum sensing. Developments in scientifc research ollow
improvements in apparatus: using tools such
Bacteriophages are used in the disinection o as the laser scanning microscope has led
water systems. researchers to deeper understanding o the
structure o bioflms.
Methods used to address pollution incidents
Responses to pollution incidents can involve bioremediation
combined with physical and chemical procedures.
When chemicals are released to the environment by accident
or through carelessness, the result can be signifcant in terms
o ecological disruption. Bioremediation is the use o microbes
to remove environmental contaminants rom water or soil.
In this section, we consider bioremediation strategies or
addressing pollutants such as benzene, petroleum oil, heavy
metals and sewage.
Not all pollution incidents can be addressed solely through
bioremediation. Bioremediation may be undesirable in the case
o heavy metals because these need to be removed rom the
ood chain. In such cases phytoremediation, which relies on
plants, might be employed. The heavy metals can bioaccumulate
in the biomass o the crop. The crop can then be incinerated to
575
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
Figure 1 Soil undergoing bioremediation concentrate the metal and then the metal can either be recycled or
at Fawley Refnery, an oil refnery properly contained.
and chemical plant located in Fawley,
Hampshire, UK There are a number o physical and chemical procedures that can be
combined with bioremediation to respond to pollution incidents.
Figure 2
Physical methods or oil spills include the use o scrubbers, detergents
Figure 3 and dispersants.
576 Chemical-contaminated soil can be removed and incinerated to
degrade volatile organic chemicals.
Soil can be removed, crushed, sited and then suspended in water
that includes chemicals that will aid in dissolving the chemicals into
the water. The chemical- contaminated water can then be puried
s e p a ra te ly.
Oxidizing chemicals such as ozone and peroxide are sometimes
injected into soils to accelerate the destruction o toxic
organic compounds.
Microorganisms have properties that make them
useful for bioremediation
Microorganisms are used in bioremediation.
Bacteria and archaeans are useul in bioremediation because they
can multiply very quickly by binary ssion and they are varied in
their metabolism. They carry out a wider range o chemical reactions,
especially inorganic reactions, than any other group o organisms. There
is oten a species o prokaryote that will perorm the necessary reaction
in a bioremediation process.
Figure 1 shows a biopile. This is a method or addressing pollution in
soil. A bulking agent such as compost, hay or other nutrient source is
dug into the piles and the piles are constantly watered. The microbial
community which fourishes digests the contaminants.
Bioremediation relies on microorganism
metabolism
Some pollutants are metabolized by microorganisms.
Microorganisms can use pollutants as energy sources, carbon sources
and electron acceptors in cellular respiration.
The bacterium Dehalococcoides ethenogenes ( shown in red in gure 2 ) has
been used to break down chlorinated solvents in soil. It uses the chlorine
compounds as electron acceptors in cellular respiration.
The bacterium Geobacter sulfurreducens uses uranium as an electron
acceptor converting it rom a soluble to an insoluble orm, which allows
the uranium to settle out and be collected.
Figure 3 shows the bacterium Acidovorax sp. ( yellow) partially coated
with iron (orange) . This bacterium is able to precipitate iron and arsenic
out o the soil and bind it. Due to this, it is being investigated as a means
o reducing the amount o arsenic present in rice elds.
B.3 environMental protection
Microorganisms can orm bioflms Figure 4 Bioflm on the bristle o a
used toothbrush
Cooperative aggregates omicroorganisms can orm bioflms.
A biolm is a colony that coats a surace as a consequence o cooperation
between individual cells. Members o a biolm colony secrete signalling
molecules that recruit independent, or planktonic, cells into the colony.
They also secrete molecules that acilitate the aggregate adhering to
the surace and acilitate individual cells sticking together. O n their
cell membranes, cells produce protein channels that acilitate the
exchange o materials with other members o the colony. While biolms
normally orm on solid suraces, they can orm on the surace o
fuids. Sometimes, they can be composed o a community o organisms
including bacteria, archaea, protozoa, algae and ungi. Dental plaque is
a biolm that can contain up to 500 taxa o microorganisms while the
biolm that orms in the lungs o patients aficted with cystic brosis is
oten composed o a single species: Pseudomonas aeruginosa.
Figure 4 shows a magnied view o a bristle rom a used toothbrush. The
surace o the bristle is covered in a biolm o cooperating bacteria. Figure 5
shows a biolm inside a catheter. A catheter is a tube used in medical
treatment to drain urine or maintain a connection to the bloodstream. The
centre part is meant to be hollow but is covered in a white-coloured biolm.
Emergent properties Figure 5 Bioflm ormed on the
inside o a catheter
Bioflms possess emergent properties.
Properties that emerge rom the interaction o the members o a
collective that are not present in the single cell orm are reerred to as
emergent properties.
In biolms, the ability o the cells to sel-organize into a complex structure
is an emergent property. Members o the colony secrete a chemical
known as exopolysaccharide (EPS) that orms into a matrix that holds the
colony together and protects it. This matrix is an emergent property.
Increased resistance to antibiotics; signalling between members o
the colony; increased virulence; the ormation o channels or water
fow inside the colony; and the ability o cells to use the matrix
to move leading to the colony itsel moving are all considered
emergent properties.
Bioflms resist antimicrobial agents
Microorganisms growing in a bioflm are highly resistant
to antimicrobial agents.
Hospital acquired inections, or nosocomial inections, are commonly
caused by biolms. Increased resistance to antibiotics sometimes occurs
in biolms and is o concern to inection control ocers within hospitals.
There are a number o proposed mechanisms or biolm antibiotic resistance.
In part, the resistance is due to the exopolysaccharide (EPS) matrix providing
a physical barrier to the entry o the antibiotic into the colony.
577
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
Antibiotics oten act on mechanisms that inhibit cell division. In some
bioflms, limited supplies o nutrients leads to a suppression o the
collective division rate which minimizes the eect antibiotics can have.
This can especially be true o individuals deeper into the colony.
Quorum sensing
Microorganisms in bioflms cooperate through
quorum sensing.
Quorum sensing is a system o behaviours that are triggered as a unction
o population density. It is observed in a diverse range o organisms.
In bacteria that orm bioflms, gene expression can be aected by
population density. S ignalling molecules released by one cell bind
to receptor molecules on another cell and lead to the expression o
genes that are likely to acilitate the development o the bioflm.
When population density is low, the concentration o the signalling
molecule is low and is insufcient to trigger coordinated behaviour.
When the population passes a threshold level; i.e., when the quorum is
achieved, the concentration o the signalling molecule reaches a critical
concentration and the behaviour becomes coordinated.
The pathogen Pseudomonas aeruginosa uses quorum sensing to coordinate
movement, EPS production, cell aggregation and the ormation o bioflms.
locally high signal
molecule concentration
EPS matrix
signal
molecule
secreted
modied
metabolism
signal molecule signal molecule
receptors secreted
relatively low concentration of
signal molecule from other cells
Free form Biolm
Figure 6
Figure 7 Bacteriophages (pink) shown infecting Using viruses to kill bacteria in water systems
a population of bacteria shown as green
Bacteriophages are used in the disinection owater systems.
When bacteria produce a bioflm, they can be difcult to eradicate. The
control o bioflms within water systems is essential.
Some o the damage that can be done includes:
Anaerobic sulphate reducing bacteria produce sulphuric acid which
can corrode pipes.
Bioflms can aect heat exchange in systems where the release o
waste heat to the environment is important.
578
B.3 environMental protection
A prolierating bioflm can reduce the diameter o a pipe. This results
in rictional drag, which lowers water pressure which leads to a need
or increased pumping power.
Bacteria can be difcult to kill when they orm a bioflm. The outer layer
o bacteria in these bioflms can be killed by disinectant, but the inner
bacteria are sheltered.
Viruses solve this problem because they spread through the entire
bioflm community. Viruses which attack bacteria are known as
bacteriophages and they are specifc to certain bacteria.
One study achieved the greatest success in killing bioflms by using a
combination o bacteriophages and chlorine. An initial treatment with
viruses ollowed by chlorine killed 97 percent o bioflms within fve
days o exposure while chlorine alone removed only 40 percent.
In addition, there may be specifc pathogenic bacteria that are living in
the bacterial community, such as coliorm bacteria. B acteriophages that
are specifc to the pathogen can be added to ensure reduction o the
particular pathogen. The T4 bacteriophage is specifc to E. coli.
Bioremediation in saline conditions
Degradation of benzene by halophilic bacteria such as Marinobacter.
The production o oil in marine environments Some archaea are adapted to living in extreme
generates volumes o saline (salty) wastewater environments such as highly saline water
that is contaminated with hydrocarbons such (fgure 9) . They are reerred to as halophiles. This
as benzene and toluene. Benzene (fgure 8) adaptation has been useul in the bioremediation
is o particular concern as it can persist in the o saline wastewater. O ne species o halophilic
environment or a long time, is moderately archaea, Marinobacter hydrocarbonoclasticus has
soluble in water and is carcinogenic; i.e., it can been shown to be able to ully degrade benzene.
lead to cancer. B ioremediation becomes difcult
in this case as the salt content may be so high
in the waste water that it kills most populations
o bacteria.
H H
C C
HC CH
CC
H = hydrogen H H
C = carbon benzene
Figure 8 Benzene molecule Figure 9 The colour in this salt pan pool is a indicator of the
presence of a population of halophilic bacteria
579
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
Bioremediation o oil spills Figure 10
Degradation o oil by Pseudomonas.
In natural environments, some petroleum seeps
through cracks and vents in the ocean foor. Some
members o the genus Pseudomonas thrive in these
communities as they can use crude oil as an energy
and carbon source. Clean-up at oil spills will oten
involve seeding the spill with Pseudomonas. These
microbes also require substances such as potassium
and urea as nutrients to metabolize the oil at a
aster rate. These nutrients are oten sprayed on
to an oil spill to aid the bacteria in their work.
Figure 1 0 shows a population o bacteria degrading
a droplet o oil suspended in water.
Bioremediation o methyl mercury
Conversion by Pseudomonas o methyl mercury into elemental mercury.
Mercury ends up in garbage dumps as a The bacterium Pseudomonas putida can convert
component o some paints and some types o the methyl mercury to methane and the
light bulbs. Elemental mercury is converted in mercury ion. Other bacteria then use the
this environment to the highly toxic organic soluble mercury ion as an electron acceptor
methyl mercury by the bacterium Desulfovibrio resulting in insoluble elemental mercury being
desulfuricans. This orm o mercury more easily reormed.
enters ood chains because it adheres to cell
membranes and can dissolve in the cell membrane. In a bioreactor, such elemental mercury can be
It can bioaccumulate within the biomass o separated rom waste water as it is insoluble and
organisms and it can biomagniy up the ood chain. will sink due to its density.
Bioflms used in trickle flter beds
Use o bioflms in trickle flter beds or sewage treatment.
The consequence o not treating sewage and adds oxygen to the sewage, which is necessary or
allowing it to fow into watercourses is nutrient the aerobic bacteria to digest the sewage content.
enrichment, or eutrophication o bodies o water.
This avours algal blooms. When the mats o
algae die, it leads to a loss o oxygen, because o
bacterial activity on the dead organic matter. This
is called biological oxygen demand.
Many sewage treatment plants make use o
biolms to address eutrophication. A trickling
lter system has a rock bed that can be up to
2 metres deep. The rocks are colonized by a
biolm o aerobic bacteria. Sewage water is
sprayed onto the rocks. The process o spraying
Figure 11
580
B.3 environMental protection
Media reports on bioflms
Evaluation o data or media reports on environmental problems caused by bioflms.
Biolms are commonly eatured in the media Most people expect to nd Salmonella
as they have a number o novel and interesting on raw meats but dont consider that it
properties. They are employed as innovative can survive on ruits, vegetables or dry
solutions to problems. At the same time products, which are not always cooked,
they have been implicated in a number o said Ponder.
environmental issues:
In moist conditions, Salmonella thrive and
Virginia Tech scientists have provided new reproduce abundantly. I thrust into a dry
evidence that biolms bacteria that adhere environment, they cease to reproduce, but turn
to suraces and build protective coatings are on genes which produce a biolm, protecting
at work in the survival o the human them rom the detrimental environment.
pathogen Salmonella.
Researchers tested the resilience o the
One out o every six Americans becomes ill Salmonella biolm by drying it and storing
rom eating contaminated ood each year, with it in dry milk powder or up to 30 days. At
over a million illnesses caused by Salmonella various points it was tested in a simulated
bacteria, according to the Centers or Disease gastrointestinal system. Salmonella survived
Control and Prevention. Finding out what this long-term storage in large numbers but
makes Salmonella resistant to antibacterial the biolm Salmonella were more resilient
measures could help curb outbreaks. than the ree-foating cells treated to the
same conditions.
Researchers aliated with the Fralin
Lie Science Institute discovered that in The bacterias stress response to the dry
addition to protecting Salmonella rom conditions also made them more likely to
heat-processing and sanitizers such as cause disease. B iolms allowed the Salmonella
bleach, biolms preserve the bacteria to survive the harsh, acidic environment o
in extremely dry conditions, and again the stomach, increasing its chances o reaching
when the bacteria are subjected to normal the intestines, where inection results in the
digestive processes. symptoms associated with ood poisoning.
O utbreaks o Salmonella associated with dried This research may help shape Food and Drug
oods such as nuts, cereals, spices, powdered Administrations regulations by highlighting
milk and pet oods have been associated with the need or better sanitation and new
over 900 illnesses in the last ve years. These strategies to reduce biolm ormation on
oods were previously thought to be sae equipment, thus hopeully decreasing the
because the dry nature o the product stops likelihood o another outbreak.
microbial growth.
Source: http://www.sciencedaily.com/releases/2013/04/
13 0 410154918. h t m
ay The development o bioflms on equipment and
piping systems in industry such as paper making
Choose one or more o the ollowing environmental acilities.
issues related to bioflms. Create a brie research report
outlining the scope o the problem. Ensure that you The development o bioflms in clean water pipes at
include the role o bioflms. Evaluate possible solutions water treatment acilities.
to the problems caused by the bioflm.
The binding o positively charged heavy metals to
The role o bioflms in increasing biological oxygen negatively charged bioflms.
demand in eutrophic bodies o water.
The sequestering o toxins within the bioflm.
581
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
Laser microscopes have enhanced our knowledge o bioflms
Developments in scientic research
ollow improvements in apparatus:
using tools such as the laser scanning
microscope has led researchers to
deeper understanding o the structure
o biolms.
Biolms have a complex structure. The position Figure 12
o individual cells in relation to one another
and the EPS matrix infuences roles and generated using a laser scanning microscope. Red
unctions. Three-dimensional visualization o dots indicate EPS, green dots indicate bacteria and
living cells serving dierent unctions can be grey dots represent host cells.
carried out using a laser-scanning microscope
in combination with dyes. This technique
allows direct observation o the biolm without
disrupting its structure.
Figure 1 2 shows an image o a ragment o biolm
extracted rom amniotic fuid. The image was
B.4 Medicine (ahl) Applications
Understanding Use o PCR to detect diferent strains o
inuenza virus.
Inection by a pathogen can be detected by
the presence o its genetic material or by its Tracking tumour cells using transerrin linked to
antigens. luminescent probes.
Predisposition to a genetic disease can be Biopharming o antithrombin.
detected through the presence o markers. Use o viral vectors in the treatment o Severe
DNA microarrays can be used to test or genetic Combined Immunodeciency (SCID).
predisposition or to diagnose the disease.
Skills
Metabolites that indicate disease can be
detected in blood and urine. Analysis o a simple microarray.
Interpretation o the results o an o ELISA
Tracking experiments are used to gain
inormation about the localization and diagnostic test.
interaction o a desired protein.
Nature o science
Biopharming uses genetically modied
animals and plants to produce proteins or Developments in scientic research ollow
therapeutic use. improvements in technology: innovation in
technology has allowed scientists to diagnose
Viral vectors can be used in gene therapy. and treat diseases.
582
B.4 Medicine (ahl)
Innovations in diagnostic techniques
Developments in scientifc research ollow improvements in technology: innovation
in technology has allowed scientists to diagnose and treat diseases.
To be useul, new methods used to diagnose a bacterial inection exists, the sample can be plated
disease must be accurate and preerably simple to on culture media to look or the growth o the
use. They should provide a result that is timely kind o bacterial colonies which characterize a
and increases the time to carry out treatment in certain disease. The limitation o this procedure is
such a way that long-term complications do not that sometimes dierent microorganisms present
result. In the case o inectious diseases, aster and in the same way. Further, some pathogens are
more accurate diagnosis can lead to treatment difcult or slow to culture.
which prevents the spread o the pathogen.
Diagnosis o genetic diseases has traditionally been
Inection by parasites has oten been diagnosed carried out by reviewing a combination o clinical
by microscopic analysis to look or the presence o observation and searching or the presence o high
the organism or evidence o its activity. levels o unusual metabolites in the urine or blood.
Diagnosis by bacterial inection has traditionally Improvements in methods o diagnosis have
been done by collecting samples o urine or stool, increased the specifcity, the speed and the
or swabs can be taken rom an inected site. I reliability o diagnosis.
High levels of metabolites can indicate disease
Metabolites that indicate disease can be detected in blood
and urine.
Inborn errors o metabolism is a term applied to a broad group o
genetically inherited disorders that aect metabolism. The majority o
these diseases are due to mutations in single genes that code or enzymes
oten resulting in a non-unctional enzyme. This results in a build-up
o substances which are toxic or a shortage o important molecules
necessary or normal unction leading to secondary symptoms. Table 1
shows three such diseases and the metabolites that are detected in blood
and urine when an individual is aected.
Newborn inants are subjected to a heel prick test to detect
phenylketonuria (PKU) , in which a blood sample is taken rom the
heel o the oot. I the child is aected, there will be elevated levels o
phenylpyruvate in the blood indicating the child lacks an enzyme or
converting the amino acid phenylalanine to tyrosine. I diagnosed quickly
enough, diet modifcation can prevent severe consequences or the child.
dss Mtbo ptwy tt s ot futog Mtbot tt s tt
LeschNyhan Production o purines Uric acid crystals in the urine
syndrome
Alkaptonuria Breakdown o the amino acid tyrosine High levels o homogentisic acid detected in both the
urine and the blood by thin layer chromatography
Zellweger Assembly operoxisomes (organelles essential and paper chromatography
syndrome or the degradation olong chain atty acids) Elevated very long chain atty acids in the blood
Table 1
583
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
Indicators of infection by a pathogen
Infection by a pathogen can be detected by the presence
of its genetic material or by its antigens.
Modern molecular methods have the advantage o being much better at
discriminating between pathogens. They can be automated to speed up
the process and they dont present the challenge o having to culture the
pathogen separately.
The Enzyme-Linked Immunosorbent Assay (ELISA) detects the
presence o antibodies to pathogens. The challenge with this diagnostic
test is that it is usually only eective once the patient has developed
an immune response to the pathogen resulting in the production o
antibodies. Recent versions o the ELISA test or the antigen directly
such as the p24 antigen rom the HIV virus.
PCR can be used to detect the genetic material o a pathogen. I primers
that have the same nucleotide sequence as the genetic material o the
The ELISA test
Interpretation of the results of an ELISA diagnostic test.
An ELISA test can be used to detect the presence linked to an enzyme. The solution is rinsed. In
o inection by a pathogen. The test works by a negative test, this would wash away the ree
testing or the presence o antibodies to the version o the capture molecule. In a positive test,
antigens o the pathogen. Alternatively, it can test they bind to the target molecule and they are not
or the antigen directly. washed away. The last step is to add the substrate o
the enzyme which changes colour when acted upon
Figure 1 shows the basis o a positive test or HIV. by the enzyme. A positive test is thereore indicated
by a coloured solution (see fgure 2) .
A capture molecule is fxed to a surace. In the
fgure, these capture molecules are antibodies to Figure 2 shows a tray o wells containing human
the HIV p24 capsid protein. blood serum rom dierent individuals being tested
or antibodies to the hepatitis C virus. Wells which
The sample to be tested is exposed to the capture remain uncoloured are negative. Those that change
surace. Because the target molecules are present in colour to yellow/orange are positive and confrm
a positive test, they bind to the capture molecules. that the patient has antibodies or hepatitis C virus.
Next a ree version o the capture molecule is
added. This version o the capture molecule is
+ +
substrate
colour change
+ by activity of
conjugated
enzyme
capture antibody detection antibody
antigen enzyme attached to detection antibody
converts substrate to coloured product
Figure 1 Steps in a positive ELISA test Figure 2 Results of multiple ELISA tests for the Hepatitis C virus
584
B.4 Medicine (ahl)
pathogen are added to a sample rom the patient, then amplication will
only occur i the genetic material o the pathogen is present.
Another way to detect the presence o a pathogen is to use DNA
probes in a microarray. These can be used to detect mRNA sequences
complementary to the pathogen in samples rom a patient.
atvty 2
Figure 3 shows a standard curve that relates quantity o 1O.D.
antigen present in the test serum to optical density, a
measure o the colour o solution. The darker the colour, 0
the higher the optical density. 0 100 200 300 400 500
antigen concentration /pg mL-1
1 Explain how the standard curve could be used. [2 ]
Figure 3
2 Determine the concentration o antigen present
at an optical density o 1.0. [1]
PCR as a diagnostic tool
Use o PCR to detect diferent strains o inuenza virus.
There are a number o clinical signs and tests that mRNA being sought was present in the original
can indicate inection by an infuenza virus. For sample and the cDNA will be amplied. A recent
some people, inection with more serious strains modication is to include fuorescent dyes into the
such as swine fu needs to be diagnosed quickly. sample that bind specically to double-stranded
This includes such patients as pregnant women, DNA. As the quantity o double-stranded DNA
elderly patients or patients whose immune system increases, fuorescence will be detected indicating
is compromised, as the inection can result in a positive test.
death. Further, some strains can produce more
serious side eects. In addition, rapid detection mRNA
can prevent a serious epidemic. The PCR test is
most likely to be able to identiy the specic strain reverse transcriptase
o the virus that inects a person. mRNA
Because the infuenza virus is an RNA virus, RNase cDNA
a variation o PCR called reverse transcription cDNA
polymerase chain reaction ( RT- PC R) is used.
Reverse transcriptase will produce a DNA primer 3
molecule rom an RNA template called cDNA.
+ Taq polymerase
The rst step involves puriying mRNA rom
cells o an inected patient. The mRNA extract Double-stranded
is converted into cDNA. Then primer sequences cDNA (target)
specic to the strain o infuenza virus being
tested or are added. I the infuenza primers bind Amplication
to sequences in the cDNA, this means that the
Figure 4
585
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
chromosome 17 chromosome 13 Genetic markers
BRCA 2 Predisposition to a genetic disease can be detected
through the presence of markers.
BRCA 1
Genetic markers are particular alleles which are associated with a
Figure 5 Chromosomal location of predisposition to having a genetic disease. They can be single nucleotide
the BRCA 1 and BRCA 2 genes polymorphisms or tandem repeats. Detection o the marker can be
achieved through such methods as PCR, and DNA proling.
Figure 6
Markers may be part o a coding or non-coding sequence; i.e., they may
contribute to the disease or they may be genetically linked to the gene
that infuences the condition. To be useul, non- coding markers need to
lie near to the deective gene to avoid being separated by crossing over.
The marker should be an allele or which the population is polymorphic;
that is, there should be a number o possible genotypes at the locus.
Researchers look or alleles which are ound more requently than
expected by chance in those people aected by the disease.
For example, mutations in the BRCA 1 and BRCA 2 genes indicate an
increased risk o breast cancer and ovarian cancer in women and the
gene itsel contributes to the onset o the cancer. The genes are ound on
chromosome 1 7 and chromosome 1 3 respectively.
There are dierent alleles o BRCA mutations. Figure 6 shows the
separation o proteins by electrophoresis. In this case, radioactively
labelled amino acids were supplied during protein synthesis and the
products were separated by electrophoresis and photographed using
lm that detects radioactivity. The arrows indicate the various types o
marker proteins produced by dierent mutations o the BRCA 1 gene.
The presence o such marker proteins in a blot rom an individual would
indicate a predisposition to cancer.
For diseases which are linked to a single gene, the marker has more
predictive power. Where diseases are strongly infuenced by the
environment or are polygenic, particular markers have less predictive
power, though considerable progress has been made recently in
establishing statistical probabilities rom more complex inheritance
patterns.
DNA microarrays
DNA microarrays can be used to test for genetic
predisposition or to diagnose the disease.
A microarray is a small surace that has a large range o DNA probe
sequences adhering to its surace. Microarrays can be used to test
or expression o a very large number o D NA sequences simultaneously.
Figure 7 A DNA microarray cartridge being The sample to be tested is the mRNA being expressed by a cell. cDNA is
loaded into a machine that will be used to ormed rom the mRNA using reverse transcriptase. At the same time
analyse the results from this test as synthesis, fuorescent dyes are linked to the cDNA. The microarray
is exposed to the cDNA sample long enough or any complementary
sequences to bind to the xed probes and then the chip is rinsed. The
chip is then exposed to laser light which will cause the fuorescent
586
B.4 Medicine (ahl)
probes to give o light where there has been hybridization between the
cDNA and the DNA probes within the chip. The brighter the light, the
higher the level o gene expression in that region.
1.28 cm A
1.28 cm
AT A A
actual size of
GeneChip array A AT C A T A T A
CA
T TC AA T A C T
C C AA C A C
A T T A T A A
A T T C TT C AT
T C G GA T C AG T
G T A G G
G AT AT T C
G A
TC AT TC
T G A TC G A
T
G T GA T G
G TG
G
non-hybridized
DNA
6.5 million locations on millions of DNA strands hybridized DNA
each GeneChip array built in each location
Figure 8 actual strand =
25 base pairs
Interpreting a microarray and then wash the chip to remove unhybridized
cDNA. The chip would then be exposed to
Analysis of a simple microarray. fuorescent light. The part o the chip where
green light is observed indicates sequences being
As an example o the use o a microarray, an expressed in the control only. The part o the chip
experimenter may want to assess the range and where there is red light is where the sequences
level o gene expression in a cancerous cell. They are being expressed by the cancerous cells only.
would extract mRNA rom control cells and Yellow light, which is a combination o green and
produce labelled cDNA rom this sample. They red light, corresponds to regions where both types
would modiy this cDNA with a green fuorescent o cells are expressing the sequence.
dye. They would then extract mRNA rom
cancerous cells, produce cDNA and label it with
red dye. They would then expose the microarray
chip to both samples, allow time or hybridization
1 Spot DNA fragments on glass 2 Isolate mRNA from cells
slide to make microarray
normal cancerous
3 Use mRNA to produce cDNA for
stability and label with dyes
Yellow: equal activity for 4 Mix and wash over microarray.
both cell types Scan with laser and detect levels
of binding/expression using
Green: higher gene activity uorescent detection
for normal cells
Red: higher gene activity
for cancer cells
Figure 9
587
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
Protein tracking experiments
Tracking experiments are used to gain inormation
about the localization and interaction o a desired
protein.
Proteins circulating in the blood can be traced i radioactive
probes are attached to them. Such tracking experiments can allow
researchers to ollow distribution and localization patterns. They can
also allow researchers to determine how the proteins interact with the
target tissue.
Radioactive atoms or molecules can be attached to the proteins and their
distribution can be tracked with PET scans.
Tracking experiments involving transferrin
Tracking tumour cells using transerrin linked to luminescent probes.
Transerrin is a molecule which binds iron. It
is taken up by more by tumour cells than by
surrounding cells.
Figure 1 0 shows a sequence o photos taken Figure 10
using luminescent dyes linked to transerrin
molecules. The experiment is being used to study
receptor-mediated endocytosis in lymphoma
cells. At zero minutes, the transerrin is shown
bound to receptors on the surace o cells. The
dots represent the fuorescent dye. The bottom
image shows some o the receptortranserrin
complexes having entered the cell. Once they
have delivered their load o iron, the receptor
transerrin complex is recycled to the cell-surace
membrane (top right) .
Biopharming
Biopharming uses genetically modifed animals and
plants to produce proteins or therapeutic use.
There are three main categories o proteins used in therapy:
antibodies, human proteins and viral or bacterial proteins (used
in vaccines) .
The production o simple human recombinant proteins or therapy,
such as insulin and growth hormone, has been most successully
carried out in genetically modied bacteria. However, the production
o more complex therapeutic proteins is more dicult to produce
588
B.4 Medicine (ahl)
in these living systems. Prokaryotic systems do not carry out the
required post-translational modifcation such as the addition o
sugars. Sometimes, only mammal cells are capable o perorming
these modifcations.
Producing these proteins in transgenic arm animals addresses the
post-translational modifcation problem. Some domestic varieties o
cows, sheep and goats have been selectively bred to produce high yields
o milk. Lactating emale animals have been engineered to secrete
recombinant proteins into their milk. The combination o these two
actors means a small herd o animals can yield a relatively large mass o
therapeutic protein.
Plant-made therapeutic proteins have been made using whole plants
and plant cell cultures. In May 201 2, the frst plant-made human
therapeutic protein was approved or use in humans by the US Food and
Drug Administration (FDA) as enzyme-replacement therapy to treat the
symptoms o Gauchers disease.
Biopharming to produce ATryn mammary isolate oocytes
gland-specic & enucleate
Biopharming of antithrombin. regulatory gene of
sequences interest transfer
Antithrombin defciency is a condition that puts reconstructed
patients at risk o blood clots during childbirth + embryo into
and surgery. ATryn is the commercial name o recipient female
antithrombin that has been produced in the
mammary glands o genetically modifed goats. target protein fuse transgenic verify presence
expression vector cell to enucleated of transgene
To achieve this genetic modifcation, the gene o oocyte
interest and specifc additional sequences have to be select
added. A specifc promoter sequence that will ensure transfect cell
that the gene is expressed in milk is necessary in cells
creating the gene construct. In addition, a signal
sequence has to be added to ensure that the protein Figure 11
is produced by ribosomes on the endoplasmic
reticulum rather than by ribosomes that are
ree in the cytoplasm. This is to ensure that the
antithrombin protein is secreted by the mammary
cells rather than released intracellularly.
Gene therapy
Use of viral vectors in gene therapy.
Some inherited diseases are caused by a deective gene, that
results in the lack o a particular enzyme or protein. Cystic fbrosis
is one such disease. It is caused by the lack o cystic fbrosis
transmembrane protein (CFTP) . This protein normally transports
chloride ions out o cells and into mucus. The chloride ions draw
589
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
retroviral vector adenoviral vector water out o the cells and make mucus watery. C ystic
DNA genome fbrosis patients suer rom thick mucus, which builds up
capsid envelope in the airways.
reverse Gene therapy may oer a cure or inherited diseases
transcriptase like cystic fbrosis. In gene therapy, working copies o
the deective gene are inserted into a persons genome.
RNA genome To do this, a gene delivery system, or vector, is needed.
Figure 1 2 shows two dierent ways o using viruses as
cell membrane vectors. The viral genome is altered so that the particles
are not virulent. The therapeutic gene is then inserted into
therapeutic the virus.
protein
Viruses that contain double-stranded ( ds) D NA, such
RNA/DNA ribosome as adenovirus, cannot cause the problems ound with
retroviruses because the viral DNA is not inserted into
nuclear nuclear the genome. However, the therapeutic gene is not
membrane pore therapeutic gene passed on to the next generation o cells, so treatment
DNA has to be repeate d more re quently. A challenge o using
viruses as vectors is that the host may develop immunity
therapeutic to the virus.
gene
The treatments described above are called somatic
therapy, because the cells being altered are somatic
(body) cells. An alternative method would be to inject
therapeutic genes into egg cells. The missing gene would
be expressed in all cells o the organism. This is called
germ line therapy.
Figure 12 Two diferent gene therapy techniques
involving viral vectors
Gene therapy to treat SCID
Use o viral vectors in the treatment o Severe Combined Immunodefciency (SCID) .
Defciency o the enzyme adenosine deaminase Removing ADA defcient lymphocytes rom
(ADA) leads to the accumulation o deoxyadenosine the patient with SCID.
within cells. This is particularly toxic to T and
B lymphocytes. The lack o unctional immune Culturing the cells in vitro.
cells leads to severe combined immunodefciency
syndrome (SCID) which is characterized by an Inecting the cultured cells with genetically
inability to fght o the simplest o inections. ADA modifed retrovirus containing the gene that
defciency was the frst condition successully treated can produce unctional ADA.
by gene therapy.
Delivering the modifed lymphocytes by
The steps involved in the successul therapy transusion back into the patient.
included:
The eect lasted or our years ater the start o
gene therapy in one patient.
590
B.5 Bioin ForM atics ( ah l)
B.5 Bfm (ahl)
Understanding Applications
Databases allow scientists easy access to Use o knockout technology in mice to
inormation. determine gene unction.
The body o data stored in databases is Discovery o genes by EST data mining.
increasing exponentially.
Skills
BLAST searches can identiy similar sequences
in diferent organisms. Explore the chromosome 21 in databases (or
example in Ensembl).
Gene unction can be studied using model
organisms with similar sequences. Use o sotware to align two proteins.
Use o sotware to construct simple cladograms
Sequence alignment sotware allows comparison
osequences rom diferent organisms. and phylograms o related organisms using
DNA sequences.
BLASTn allows nucleotide sequence alignment
while BLASTp allows protein alignment. Nature of science
Databases can be searched to compare newly Cooperation and collaboration between groups
identied sequences with sequences o known o scientists: databases on the internet allow
unction in other organisms. scientists ree access to inormation.
Multiple sequence alignment is used in the
study o phylogenetics.
EST is an expressed sequence tag which can be
used to identiy potential genes.
The role of databases in genetic research
Databases allow scientists easy access to inormation.
A database is a structured collection of information stored on a
computer. It can include data in a range of formats including qualitative
information, articles, images or quantitative information.
Types of databases used in bioinformatics include:
Nucleotide sequence databases such as EMBL (The European
Molecular Biology Laboratory) .
Protein sequence databases such as SwissProt.
Three-dimensional structure databases such as PDB (Protein Data Bank) .
Microarray databases such as ArrayExpress which contain
information about the level and types of mRNA expressed in
different cells.
Pathway databases which contain information about enzymes and
reactions and can be used to model metabolic pathways. An example of
such a database is KEGG (Kyoto Encyclopedia of Gene and Genomes) .
591
B BIOTECH NOLOGY AN D BIOIN FORM ATICS
toK Hypothesis testing is increasingly possible by extracting data rom a database
rather than the researcher collecting the data directly or themselves.
t wh exen des scenfc reserch
requre reguln? i des requre A researcher can employ a database to do a number o tasks:
reguln wh shuld dmnser he
regulns? add the results o their research or others to access
In 1999 a patient died as a result o extract subsets o data
participation in clinical trials or gene therapy.
He sufered rom ornithine transcarbamylase query the database by searching or a particular piece o data.
deciency, or OTC, a liver disease marked
by an inability to metabolize ammonia. Growth in information housed in databases
Ammonia is a waste product oamino acid
metabolism. He had been able to survive up The body o data stored in databases is increasing
to that point because odietary modication exponentially.
and medication. The trial he participated in
involved being injected with adenoviruses Advances in technology have meant that the rate o creation and
carrying the gene or transcarbamylase. He publication o data is increasing. Advances in genome sequencing
died within days due to a strong immune technology, microarrays, 3 -D modelling programmes and computing
response to the viral vector. An investigation power have resulted in a number o large-scale collaborative research
concluded that the scientists involved in the projects which have generated an exponential growth in data housed
trial violated several rules oconduct. in databases. One research report tracked the growth in inormation in
bioinormatics databases and concluded that it has a doubling time o
Four other patients who had received between 1 2 and 24 months.
the treatment had reactions that were
deemed so severe that the trial should access nrmn ssues n bnrmcs
have ended.
Cooperation and collaboration between groups o
The inormed consent orms did not scientists: databases on the internet allow scientists ree
include inormation about primates access to inormation.
that had died in similar trials.
Most people presume that collaboration and cooperation between
The patient had levels o ammonia researchers characterizes the scientifc endeavour. Most o the
that were so high he should have been important bioinormatics databases are public and reely accessible
excluded rom the study. to all researchers. Oten, once data is added to one database, it is
immediately synchronized with data in other databases. Such open
A principal investigator o the study access and synchronization acilitates collaboration and a spirit
had a major interest in the outcome o o cooperation.
the trial as he held patents on the OTC
treatment. One view is that the commercialization o bioinormatics databases is a
threat to this spirit.
From Welcome to the Genome by Bob De
Salle and Michael Yudell Some researchers working in private companies do not post their
sequence inormation because o the need to make a proft. Some
1 Explain what is meant by inormed databases that have been public in the past have been taken over by
consent. or-proft companies who have started to charge or access to sequence
inormation. Two examples are the Saccharomyces cerevisiae ( yeast) and
2 ) Suggest what policy instruments Caenorhabditis elegans ( soil roundworm) databases, two o the most
might be put in to place to prevent widely studied eukaryote model organisms. This was controversial as
such occurrences. some o the inormation in the databases was derived rom published
studies and personal communications.
b) Who should administer these
policies governments, other The academic j ournal Science twice created controversy due to the
scientists or research institutions? competing imperatives o public and private science. In 2001 , the
journal published the company Celeras version o the sequence o
592
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