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Unit 5 - Control in cells and in organisms Responses A taxis is a simple response to stimuli determined by the direction of the stimulus y
Positive phototaxis means the organism moves towards light
y
Negative
chemotaxis means the organism moves away from chemicals
A kinesis is a non-direc n on-directional tional response where an organism increases the rate of change of movement in response to
negative stimuli and decreases the rate of change of movement in response to positive stimuli. This ensures that the organism has a higher chance of remaining in f avourable avourable conditions. A tropism is a growth movement of plants in response to directional stimuli. y
Positive phototropism is exhibited by leaves growing towards light
y
Positive hydrotropism is exhibited by roots growing towards water
y
Negative
phototropism is exhibited by roots growing away from light
The nervous system has several subdivisions that can be further subdivided. 1. The central nervous system consists of the brain and the spinal cord. 2. The The peripheral nervous system consists of pairs of nerves that originate from the brain or spinal cord and are subdivided into: a.
Sensory neurones,
b.
Motor
that carry impulses from receptors to the central nervous system
neurones , that carry impulses from the central nervous system to effectors and can be
subdivided further into: i. The voluntary nervous system that carries nerve impulses to muscles under conscious control ii. The autonomic nervous system that carries nerve impulses to glands, smooth muscle and cardiac muscle and not under conscious control Reflex
arcs are important because:
y
They are involuntary, so do not require the brain to process information
y
They protect the body from harmful stimuli and are present from birth
y
They are fast as the neurone pathway is short
An example reflex arc is the withdrawal withdrawal of the hand from a heat stimulus:
1. A stimulus the heat from the hot object on the hand 2. A receptor receptor temperature receptors in the skin create a nerve impulse in the sensory neurone 3. A sensory neurone passes the nerve impulses from the receptor to the spinal cord 4. An intermediate neurone links the sensory neurone to the motor neurone in the spinal cord 5. A motor neurone carries the impulse from the spinal cord to the muscle 6. An effect effect the muscle contracts due to the nerve impulse 7. A response the hand moves away from the hot object
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Control
of Heart Rate
The autonomic nervous system is responsible for the control of the heart rate. It controls the involuntary actions of internal muscles and glands with two divisions: y
The sympathetic nervous system that stimulates effectors
y
The parasympathetic nervous system that inhibits effectors
Heart rate is controlled by the medulla oblongata is divided into two, a centre that increases heart rate and a centre that decrease heart rate, both of which are connected to the sinoatrial node. It responds to two types or receptors , chemoreceptors that detect chemical changes and baroreceptors that detect pressure changes.
Control by chemoreceptors 1. Chemoreceptors in the aorta and in the walls of the carotid artery detect a lowered pH in the blood , caused by a high carbon dioxide concentration in the blood. 2. The chemoreceptors increases the frequency of nerve impulses to the centre of the medulla oblongata that increases heart rate 3. The centre increases the frequency of impulses via sympathetic nervous system to the sinoatrial node which increases heart rate 4. This causes an increase in blood flow to the lungs which decreases carbon dioxide concentration and so lowers pH 5. Chemoreceptors detect the change and lowers the frequency of impulses to the medulla oblongata which in turn reduces the frequency of impulses to the sinoatrial node via the sympathetic nervous system
Control by baroreceptors y
When blood pressure rises, heart rate decreases
y
When blood pressure falls, heart rate increases
The P acinian corpuscle responds to mechanical stimuli such as pressure. It is an exposed neurone ending surrounded by layers of connective tissue. I t functions as follows: 1. In the resting state stretch-mediated sodium channels on the cell membrane are closed and the cell maintains a resting potential 2. When pressure is applied the stretch-mediate sodium channels open and sodium ions diffuse into the neurone 3. The influx of sodium ions causes the cell to depolarise. If the threshold potential is reached a generator potential is generated and creates an action p otential that passes along the neurone. Rod
cells are a type of receptor in the eye that cannot distinguish wavelengths of light so produces a black and white
image. They are more common than cones and are mostly found at the peripheries of the retina. y
They are very sensitive because many rod cells connect to a single bipolar neurone (retinal convergence) and can combine several small s timuli to overcome the threshold potential and create a generator potential.
y
They have low visual acuity due to the fact that several rods are connected to a single bipolar so no matter how many of the neurones are stimulated only one action potential is ever generated and so the separate sources of light that stimulate each neurone cannot be distinguished.
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C one
cells are receptors in the eye and exist in three varieties, each corresponding to a different wavelength of l ight.
A range of colours can be perceived due to different combinations of cone cells being stimulated. There are fewer
cone cells than rods and are concentrated at the fovea. y
They are not very sensitive because each cone cell is connected to a single bipolar neurone so a single cone cell must overcome the threshold potential for a generator potential on its own.
y
They have a high visual acuity due to the fact that a single cone is connected to a single bipolar neurone so each source of light that affects a cone can be distinguished.
Coordination H ormonal
System Communication by chemicals called h ormones Transmitted relatively slowly by the blood system throughout the body, but only target organs respond Widespread, slow and long-lasting response Effect may be permanent and irreversible Chemical mediators
Nervous System Communication by nerve impulses Transmitted rapidly by neurones to specific parts of the body Localised, rapid and short-lived response Effect is temporary and reversible
are important at the cellular level and are produced by mammalian cells. They affect cells in
their immediate vicinity. Histamines are an example and they dilate arteries and arterioles and make capillaries more permeable. Prostaglandins have the same affect but also affect blood pressure and n eurotransmitters.
P lant growt h
factors exert their influence by affecting growth and are essential for plants to be able to respond to
changes in both their internal and external environments. An example is indoleacetic acid (IAA) which causes plant cells to elongate. y
Cells in the shoot produce IAA which is then transported down the shoot.
y
The IAA is initially transported to all sides as it begins to move down the shoot.
y
Light causes movement of IAA towards the shaded side, causing the concentration of IAA to build up more on the shaded side than the light side.
y
IAA causes the cells to elongate more on the shaded side, meaning that side grows faster and so the shoot bends towards the light.
Neurones are specialised cells that carry nerve impulses. They are made out of: y
A cell body which contains a nucleus.
y
Dendrons
y
An axon that is a single long fibre that carries nerve impulses away from the cell body
y
Schwann cells
y
Myelin
which subdivide into dendrites that carry nerve impulses towards the cell body that surround the axon and provides electrical insulation
sheaths are insulating membranes surrounding the axon made from Schwann cells and contain
myelin. Myelinated neurones have a myelin sheath, unmyelinated neurones lack myelin sheaths. y
Nodes
of Ranvier are the gap between adjacent myelin sheaths.
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A nerve impulse is a wave of depolarisation along the axon membrane. It is the reversal of the resting potential. The +
+
resting potential is maintained by sodium-potassium pumps that pump 3 x Na out of the cell and 2 x K into the cell , a few potassium ion channels are also open at rest and allow potassium ions to diffuse back out of the axon. Since the movement of positive ions outwards is greater than the movement of positive ions inwards a p otential difference of around -65mV is maintained and the axon membrane is polarised . The
Action Potential
1. A stimulus causes sodium voltage-gated channels in the axon membrane to open and allow sodium ions to diffuse into the axon down the electrochemical gradient. They trigger a reversal in the potential difference of the membrane and as more sodium ions diffuse into the axon more sodium ions open to depolarise the axon. 2. At around +40mV the sodium voltage-gated channels close and potassium voltage-gated channels begin to open. Potassium ions diffusing out of the cell causes more potassium channels to open and the diffusing out of potassium ions causing the axon to repolarise. 3. The outward diffusion of potassium causes the potential difference of the axon membrane to become more negative than at resting potential and is hyperpolarised which is also known as the refractory period . 4. The resting potential is restored by sodium-potassium pumps as the potassium voltage-gated channels close and the cell is repolarised .
Propagation of an action potential 1. In an unmyelinated axon , once a stimulus causes a sudden influx of sodium ions the axon membrane is depolarised. 2. This establishes localised electrical circuits that cause sodium voltage-gated channels to open further along the membrane and cause d epolarisation in the new region. Behind this new area potassium ions begin to leave the axon. 3. The action potential continues to propagate along the axon and the original region is repolarised . After a short while the original region of depolarisation is back at resting potential. In myelinated axons the insulating myelin sheath prevents action potentials forming except at the nodes of Ranvier . The localised electrical circuits therefore arise only at the nodes of Ranvier and the action potential jumps from node to node and so is much faster than in unmyelinated axons. This is called sala33tatory conduction.
Properties of t he nerve impulse The speed of action potentials varies on several factors. y
The myelin sheath a myelin sheath allows action potentials to jump between nodes of Ranvier and so travel faster.
y
The diameter of the axon the greater the diameter of the axon the faster the speed of conductance
y
The temperature this affects the rate of diffusion of the ions involved in the action potential
The refractory period serves three purposes: y
Ensures nerve impulses are unidirectional because an action potential cannot be triggered in a region of the axon that is hyperpolarised
y
Ensures discrete nerve impulses as a new action potential cannot be formed immediately after the first due to the hyperpolarised region
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The all-or-not hing principle is that below the threshold value an action potential is never generated and above the threshold value an action potential of the same si ze is always generated. This means that it doesnt matter how strong a stimulus is in regards to the size of an action potential. Size of the stimulus can be determined by the number of impulses and by having different neurones with different threshold values.
Synapses A synapse is the point where one neurone connects to either another neurone or an affecter. A synapse consists of a
postsynaptic membrane which has sodium channels on its surface, the synaptic cleft (the gap between the two neurones) and the presynaptic knob which contains vesicles filled with neurotransmitter, large amounts of mitochondria and calcium ion channels. y
Synapses are unidirectional because only the postsynaptic neurone have receptors on them
y
Spatial summation is where several presynaptic neurones release neurotransmitter simultaneously in order to exceed the threshold potential
y
T emporal summation is where a single presynaptic neurone releases neurotransmitter repeatedly over a
short amount of time to exceed the threshold potential y
I nhibition is due to postsynaptic membranes having chloride channels that certain neurotransmitters can
open and further polarise the cell Transmission
across a cholinergic synapse and at neuromuscular junctions
A cholinergic synapse is a synapse that uses the neurotransmitter acetylcholine. They occur in the central nervous
system and at neuromuscular junctions. 1. When an action potential arrives at the presynaptic knob it causes calcium ion gates to open and so calcium ions diffuse into the presynaptic knob. 2. This causes vesicles to fuse wi th the presynaptic membrane and release acetylcholine into the synaptic cleft 3. The acetylcholine diffuses across the synaptic clef t and binds with receptors on the postsynaptic membrane that causes sodium ion channels to open, sodium ions to diffuse into the membrane and polarise the postsynaptic membrane and generate an action potential. 4. Acetycholineesterase then hydrolyses acetylcholine into acetyl and choline closing the sodium channels. The acetyl and choline then diffuse back i nto the presynaptic knob where ATP is used to recombine acetyl and choline into acetylcholine.
Muscle Sk eletal
contraction
muscle is made out of muscle fibres, each of which is an individual cell with shared nuclei and a shared
sarcoplasm (cytoplasm in muscles). Each muscle fibre is made up of several myofibrils; myofibrils are made up of two types of muscle fibre. Myosin is a thick long rod-shaped fibre with a bulbous head that projects out to the side. Actin is a thinner fibre and consists of two strands twisted around one another. Myofibrils appear striped due to their alternating light-coloured and dark coloured bands. The lighter areas are called isotropic bands (I-bands) and are the region where actin and myosin fibres do not overlap. The darker areas are call ed anisotropic bands (A-bands) and are the region where actin and myosin fibres do overlap. At the centre of each A-band is a lighter H-zone where there is only myosin. At the centre of each I-band is the Z-line. The distance between two Z-lines is the sarcomere. When a muscle contracts the sarcomeres shorten.
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There are two major types of muscle fibre: y
Slow twitc h muscle fibres contract more slowly and provide less force but do so over a longer period. They have:
y
o
A large store of myoglobin (an oxygen storing molecule)
o
Plenty of blood vessels to deliver oxygen and glucose
o
Large amounts of mitochondria
o
Lots of glycogen
F ast twitc h muscle fibres contract faster and provide more force over a sh ort period only. They have: o
Thicker and more numerous myosin filaments
o
A high concentration of glycolysis enzymes
o
A store of phosphocreatine. Phosphocreatine is a molecule that can rapidly and anaerobically
phosphorylate ADP to ATP. It acts as a reserve supply of phosphate and is replenished from ATP once the muscle has relaxed. The sliding filament mec hanism is how muscles contract. When a muscle contract the I-band becomes narrower, the Z-lines move together and the H-zone shrinks or disappears and the A-band remains around the same size. This is because when muscles contract it is the actin filaments being pulled into the centre by myosin and sliding over each other by the following mechanism: 1. The action potential on the muscle fibre travels through tubules in contact with the sarcoplasmic reticulum of the muscle and causes the sarcoplasmic reticulum to release calcium ions Ca2+. 2. The calcium binds to the tropomyosin. Normally the tropomyosin physically obstructs binding sites for crossbridge; once calcium binds to the tropomyosin it move out of the way , unblocking the binding sites. The calcium ions also activate the enzyme ATPase. 3. The myosin heads already have an ADP molecule attached so is capable of binding to the actin filament at binding sites and forming cross-bridges . 4. The myosin heads then changes their angle , pulling the actin filament along and releasing the ADP molecule. An ATP molecule then attaches to the myosin head detaching it from the actin filament.
5. The ATPase enzyme then hydrolyses the ATP molecule and provides energy for the myosin heads to change back to its original angle. 6. With the ADP attached the myosin head binds to a binding site further along the actin filament. 7. This process continues until nervous stimulation ceases, at which point Ca
2+
is actively transported back into
the sarcoplasmic reticulum and tropomyosin once again blocks the binding sites.
Homeostasis Homeostasis is importance in the maintenance of a constant internal environment . Organisms with the ability to maintain a constant internal environment has a greater chance of survival due to being more independent of their environment.
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Temperature Regulation
Enzymes are temperature sensitive and small fluctuations can seriously impair enzyme activity, maintaining a constant internal temperature means biochemical reactions can continue at a steady rate. Mechanisms of heat loss include evaporation of water and the loss of heat by conduction, convection and radiation. Mechanisms of heat gain include production of heat by metabolism and the gain of heat from the environment by conduction, convection and radiation. C onduction is the transfer of energy through matter in solids, convection is the transfer of heat due the movement of warned matter and radiation is the transfer of heat by electromagnetic waves. In ectot herms most heat is gained from the environment so their body temperature can fluctuate. They use behavioural mechanisms to maintain a constant internal temperature. In endot herms physiological mechanisms are also used in the maintenance of the internal body temperature. Thermoreceptors in the blood detect changes to core temperature and thermoreceptors at the skin detect changes to surface temperature. Endotherms have the hypothalamus i n the brain as the coordinator of the regulation of body temperature, it is divided into the heat loss and the heat gain centres. Heat is gained and conserved through a variety of mechanisms: y
V asoconstriction
the diameter of arterioles near the surface of the skin is made smaller and this results in
less blood reaching the skin surface through capillaries so less heat is l oss through radiation Shivering rhythmic muscle contractions produce metabolic heat y
Raising of hair the hair traps a layer of insulating air by the skin and so conserves in heat in furry animals
y
Increase in metabolic rate hormones that increase metabolic rate are secreted so more heat is generated
Heat is lost through a variety of mechanisms: y
V asodilation
the diameter of the arterioles near the surface of the skin is increased and this results in more
blood reaching the skin surface and radiating away y
Increase sweating water evaporates from the skin surface and takes heat with it. In furry animals cooling is achieved by panting instead.
y
Blood
Lowering
of hair the hair traps a thinner layer of air so less heat is conserved
glucose regulation
Blood glucose affects the water potential of cells , therefore it is essential to keep a constant water potential in order to prevent cells shrinking or b ursting. Constant blood glucose also supplies a constant supply of substrate for respiration. Blood glucose is controlled mainly by two hormones that act antagonistically glucagon and insulin. I nsulin is secreted by the -cells of the islets of langerhans in the pancreas when blood glucose levels rise. It binds to
glycoprotein receptors on cell-surface membranes and reduces blood glucose levels by: y
Increasing the rate of absorption of glucose into cells
y
Increasing the respiratory rate of cells , and so increasing glucose uptake
y
Increasing the rate of the conversion of glucose into glycogen glycogenesis
y
Increasing the rate of the conversion of glucose into fat
These effects lower blood glucose, which is then detected by the -cells and so less insulin is secreted.
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Glucagon is secreted by the -cells of the islets of langerhans in the p ancreas when blood glucose levels fall. It binds
to receptors on liver cells only and l ower blood glucose levels by: y
Increasing the rate of conversion of glycogen to glucose glycogenolysis
y
Increasing the rate of conversion of glycerol and proteins into glucose gluconeogenesis
Adrenaline is also involved in the regulating of blood glucose levels and is secreted by adrenal glands above the
kidneys during times of excitement. Adrenaline increases blood glucose levels by: y
Activating an enzyme to convert glycogen to glucose glycogenolysis
y
Deactivating the enzyme for synthesising glycogen from glucose - glycogenesis
Being proteins these hormones function by the second messenger model 1. The hormone binds to a receptor site on the cell-surface me mbrane 2. This activates an enzyme inside the cell membrane that converts ATP to cyclic ATP (cAMP). This is the second messenger and activates other enzymes that will achieve the desired effect (such as glycogenolysis) Diabetes y
Type 1 diabetes is due to the body being unable to produce insulin a nd begins early. It may be caused by an autoimmune reaction against the -cells of the islets of the langerhans. It is generally controlled by injections of insulin.
y
Type 2 diabetes is due to the glycoprotein receptors being unresponsive to insulin. It generally occurs in people over the age of 40, although has a higher risk of occurring in people with poor diet. It is generally controlled by diet and occasionally by drugs.
Feedback Negative feedback is where corrective measures result in corrective measures to be turned off. Separate feedback mechanisms give a great degr ee of control P ositive feedback is where corrective measures result in
corrective measures continuing to operate. It is rare in
biological systems, but an example would be depolarisation. The oestrous cycle is an example of a negative feedback system and is regulated by four hormones. LH and F SH produced in the pituitary gland and progesterone and oestrogen produced in the ovaries. In humans and some primates the shedding of the lining occurs as part of the cycle so is called themenstrual cycle. 1.
FSH
is produced from the beginning of the menstrual cycle even as the uterus lining is shed (Days 1-5). FSH
stimulates the maturation of follicles. 2. The growing follicles secrete a small amount of oestrogen which inhibits FSH and LH release. 3. As the follicles continue to grow more and more oestrogen is produced until around day 10 it begins to stimulate FSH and LH release 4. The surge in LH causes one of the follicles to release its egg in a process known as ovulation. LH also causes the follicle to mature into a corpus luteum which secretes progesterone and some oestrogen. 5. The progesterone inhibits the release of FSH and LH and also maintains the uterus lining. 6. If the egg is not fertilised the corpus luteum breaks down, progesterone is no longer produced and the uterus lining is shed as FSH production starts up again due to a lack of progesterone and the cycle repeats.
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Genetics
& Protein Synt hesis
y
Base
triplets in DNA code for specific amino acids
y
The code is degenerate as many triplets can code for the same amino acid
y
The code is universal in that almost all organisms the same triplet will code for the same amino acid
y
The code is non-overlapping as each base in a sequence is read only once
y
DN A is a double polynucleotide chain made with pentose deoxyribose sugar and a double helix structure. It
is the most stable of DNA, mRNA and tRNA as it is the hereditary molecule. y
mRN A is a single polynucleotide chain made with pentose ribose sugar, uracil as a base instead of thymine and a single helix structure. It is the least stable of DNA, mRNA and tRNA. A triplet is a codon.
y
t RN A is a single polynucleotide chain made with pentose ribose sugar, uracil as a base instead of thymine and a folded clover shaped structure. It is more stable than mRN A but less stable than DNA. A triplet is an anticodon.
DNA is contained inside the nucleus, ribosomes outside. In order for proteins to be synthesised mRNA must be
created to transfer the genetic information to the ribosomes. This process is known as transcription. 1.
DN A
helicase breaks the hydrogen bonds between strands of DNA causing the strands to separate
2. The enzyme RN A polymerase moves along the template strand of the DNA causing RNA nucleotides to line up in complementary pairs. Guanine on the DNA links with cytosine similarly cytosine links with guanine and thymine links with adenine. The only exception is adenine which links with uracil. 3. The RNA polymerase links RNA nucleotides with each other one at a time and moves along the DNA strand until the stop triplet at which point the RNA polymerase and pre-mRNA detaches a.
In eukaryotes the pre-mRNA is converted into mRNA by having non-coding introns removed and leaving behind only coding exons. This process is called splicing.
4. The RNA then leaves the nucleus via a nuclear pore in order to begin translation. T ranslation is the conversion of the genetic code contained in mRNA into a polypeptide chain and is done by
ribosomes. 1. A ribosome attaches to an mRN A molecule. 2. A tRNA molecule with a complementary anticodon sequence to the first codon carries a specific amino acid and pairs up with the first codon on the mRN A. 3. A tRNA molecule with a complementary anticodon sequence to the second codon carries a specific amino acid and pairs up with the second codon on the mRNA. 4. By means of an enzyme and ATP the two amino acids are joined by a peptide bond as the ribosome moves along the mRNA by one codon. 5. As the first tRNA molecule leaves a third tRNA molecule arrives with an amino acid and a complementary anticodon. 6. The ribosome moves along the mRNA until the stop codon is reached at which point the ribosome and the last tRNA molecule detaches from mRNA and the polypeptide chain is complete. The polypeptide chain will then be modified to create a functional protein.
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Gene y
mutations In a deletion mutation a one base is missed out. This is called a frameshift and often results in a nonfunctional protein.
y
In a substitution mutation one base has been replaced by another. There are several outcomes of this: o
A nonsense mutation is where a substitution results in a stop code, radically altering the protein
o
A missense mutation is where a substitution results in a diff erent amino acids which can have a
range of effects from a functional protein to a non-functional protein o
A silent mutation is where a substitution results in the same amino acid due to the degenerate
nature of the genetic code. This has no effect. Cell division is controlled by two types of genes proto-oncogenes and tumour-suppressor genes. y
P roto-oncogenes
stimulate cell division. In a normal cell, growth factors attach to receptor proteins and
trigger relay proteins to activate cell division. When a mutation occurs oncogenes may be formed, with oncogenes cell division is overstimulated either due to the rec eptor protein being permanently on or an excess of growth factors y
T umour-suppressor genes inhibit cell division. If a mutation causes the gene to become in activated, the rate
of cell division increases and if the cells do not die they can result in tumours. Gene
expression
A fertilised egg is a totipotent cell, this means it can differentiate into any type of cell. When cells specialise only
parts of their DNA is translated. Plants retain totipotent cells into adulthood and so a full plant can be grown in vitro. Only embryos are totipotent in mammals. In adults some cells are multipotent , meaning they can develop into a few other types of cells, such as bone marrow. It is hoped human embryonic stem cells may be used medically to grow new tissue for transplant without risk of rejection. Oestrogen is a hormone and works by a different mechanism to protein hormones. 1. Oestrogen is lipid soluble so diffuses easily across the cell-surface membrane 2. An inhibitor molecule blocks the DN A binding site of the transcriptional factor 3. When oestrogen binds to the receptor on the transcriptional factor it causes a change in shape that releases the inhibitor 4. The transcriptional factor can now bind with DNA and begin the process of transcription Small interfering RN A (si RN A) is a small double stranded section of RNA with a number of potential uses including blocking genes involved in genetic diseases and studying biological pathways by removing a gene to see the role of the blocked gene. siRNA operates as follows: 1. An enzyme cuts up larger double-stranded molecules of RNA into smaller sections called siRNA 2. One of the two strands will combine with a different enzyme 3. The siRNA strand will pair with a mRN A strand with a complementary base sequence 4. The enzyme attached to the siRNA will cut up the mRNA into small pieces incapable of being translated, preventing protein synthesis
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DN A Technology The DNA of two different organisms that have been combined is known as recombinant DN A. Any resulting organism is known as a genetically modified organism (GMO). Reverse transcriptase
is an enzyme used to obtain DNA fragments without any non-coding introns.
1. A cell that produces the desired protein, and therefore has a large amount of the desired mRNA, is isolated. mRNA is then extracted from the cell. 2. Reverse transcriptase uses the mRNA as a template to produce a complementary DN A (cDN A strand) 3. A DNA double helix can then be produced by using DNA polymerase on the cDNA strand. Restriction endonucleases
are enzymes that cut up DNA. Some do a straight cut leaving blunt ends. Others have a
staggered cut and leave stick y ends. Each restriction endonuclease has a different recognition sequence. An example is the AAGCTT 6bp palindromic sequence of the HindIII restriction endonuclease. I n vivo gene cloning is where genes are cloned inside organisms.
1. A desired
DN A
fragment and a plasmid with a mar k er gene are cut using the same restriction endonuclease
enzyme at the marker gene to leave sticky ends and deactivate that particular marker gene in the plasmid. 2.
The vector and DNA fragment are combined at the sticky ends by an enzyme called DN A Ligase.
3. The vector is then introduced into bacterial cells in a process called transformation. However many cells will not take up the vector or take up an unchanged vector. 4. The transformed bacteria are identified by a variety of methods all involving mar k er genes. A sample of the bacteria is placed in a nutrient agar with the antibiotic whose antibiotic resistance gene is present in the plasmid. Any survivors have taken up the plasmid and its antibiotic resistance gene. Then to identify bacteria with transformed plasmids the following methods are used: a. Antibiotic resistance mar k ers i. A replica plate is made in a n utrient agar with the antibiotic whose antibiotic resistance gene that has been cut up by the restriction endonuclease to insert the DNA fragment. Any colonies that die in this p late have been transformed and the colonies in the same position on the first plate are isolated. b. Fluorescent mar k ers i. A plasmid with a green fluorescent protein producing gene is used, the DN A fragment being inserted into this gene deactivating it. ii. Transformed bacteria can then be identified by viewing under a microscope as transformed genes do not fluoresce c.
Enzyme mar k ers i. The enzyme lactase is produced by the marker gene i n the plasmid and changes the colour of a colourless substrate blue. ii. Transformed bacteria can be i dentified by finding bacteria that do not change the colour of the colourless substrate
5. Transformed bacteria are then cloned to produce the product in a large scale.
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I n vitro gene cloning is done by the polymerase chain reaction (PCR). To carry out PCR the process requires the
following: y
The desired DN A fragment
y
Thermostable DN A polymerase
y
Primers short sequences of nucleotides complementary to each end of the two DNA fragments
y
Nucleotides
y
Thermocycler a computer controlled machine that varies temperature over time
- all four bases
The polymerase c hain reaction is carried out in three stages repeatedly cyclically, each stage doubling the number of DNA strands:
1. The reaction mixture is heated to 95C and so break s the hydrogen bonds between the two DNA strands, separating them. o
1. The reaction mixture is then cooled to 55 C causing the primers to join to the complementary bases at each end of the DNA strands. This serves the purpose of preventing the strands rejoining and also to provide starting sequences for DNA polymerase 2. The reaction mixture is then heated up to 72oC as this is the optimum temperature for the
DN A polymerase
enzyme to function. The enzyme adds complementary nucleotides along each strand, starting at the primers. I n vitro The process is extremely rapid, meaning a tiny sample can be multiplied to many millions in hours that would take in vivo days The process does not require living cells, only DNA fragments 20% rate of inaccurate copies Takes an entire strand Only produces DNA
A Recombinant DN
I n vivo Useful for introducing genes into different organisms to produce GMOs. Also useful in gene therapy
No risk of contamination as restriction endonuclease will only cut a specific sequence Very accurate cloning Cuts out a specific gene Transformed bacteria can produce gene products
has many potential uses.
y
Increase the yield from animals or plant crops
y
Improve the nutritional content of meals
y
Introduce resistance to disease and pests
y
Make crops tolerant to herbicides
y
Develop tolerance for extreme conditions
y
Making vaccines
y
Producing medicines for treating disease
Gene therapy is to cure defective genes using healthy genes, an example of a genetic disease being cystic fibrosis. Cystic fibrosis is a disease caused by a mutant recessive allele that has a deletion mutation. The mutation causes a
chloride channel called CTFR (cystic fibrosis trans-membrane-conductance-regulator) to not function so chloride ions stay trapped in epithelial cells. This results in an osmotic gradient not being set up between the inside of the cell and the outside in epithelial cells. This leads to mucus being viscous and sticky which can lead to mucus congestion in the lungs causing breathing difficulties , accumulation of mucus in pa ncreatic ducts leading to the formation of fibrous cysts and finally accumulation of mucus in the sperm ducts possibly leading to infertility.
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y
Gene replacement is where the defective gene is replaced by a health gene
y
Gene supplementation is where a dominant health gene is inserted alongside a defective recessive gene
y
Germ line therapy is where gene therapy occurs in fertilise egg cells ensuring all future offspring do not have the disease. This is currently illegal
y
Somatic-cell
therapy is where gene therapy only targets the affected tissue and tends to need to be
reapplied occasionally; offspring are also capable of inheriting the disease. Adenoviruses can be used as a vector to deliver the CTFR gene into epithelial cells.
1.
The adenoviruses are made harmless by interfering with a gene involved in replication
2.
The adenoviruses are then grown in laboratory epithelial cells along with plasmids that contain the CTFR gene. The viruses incorporate the CTFR gene and then are isolated from the epithelial cells.
3.
The adenoviruses are then introduced into the nostrils of patients where they inject their DNA, including the CTFR gene, into the epithelial cells of the lungs
Liposomes,
lipid molecules, can also be used as a vector to deliver the CTFR gene into epithelial cells.
1.
CTFR genes are isolated from human tissue an d inserted into plasmid vectors from bacteria
2.
Plasmid vectors reintroduced into bacteria for cloning
3.
Plasmids then removed from bacteria again and inserted into liposomes
4.
The liposomes are sprayed as an aerosol into the lungs
5.
The liposomes pass across the phospholipid bilayer
These forms of delivery can be ineffective because: y
Adenoviruses may cause infections
y
Patients may become immune to adenoviruses
y
Liposome aerosol may not pass through tiny bronchioles in lungs
y
Very few CTFR genes are actually expressed
DN A probes are short, single-stranded sections of DNA with a label. Common labels are radioactivity and
fluorescence. They are used to identify the presence of certain sections of DNA, they achieve this by binding to complementary bases on one of the strands in the process known as
DN A
hybridisation.
Gel electrophoresis is a method of separating DN A fragments by size (strand length).
1. DN A fragments are placed in agar gel and voltage applied across it, the negatively charged DNA is attracted to the positive anode 2. The fragments are attracted the anode but travel at different speeds as the different sizes create different resistances 3. The smallest fragment is at the bottom and the rest in increasing height. 4. A sheet of photographic f ilm can be placed over the agar gel in order to expose the film and show where each DNA fragment is Restriction mapping
is done using restriction endonucleases. Several enzymes are used together to create a
restriction map of a DNA fragment. The distance between recognition sites can b e used to find the size of fragments and the type of enzyme used for each cut on the restriction map.
14 | P a g e
DN A
sequencing can be done using the c hain terminator tec hnique or t he Sanger met hod . This requires a group of
four test tubes, each containing the single-stranded fragments of DNA to be sequenced, DNA nucleotides , one of four terminator nucleotides per test tube, a labelled primer and DNA polymerase. 1. As the binding of nucleotides is a random process, the likelihood of a terminator or normal nucleotide binding is equal. 2. When a terminator nucleotide binds the particular DNA strand cannot continue to be synthesised . As a result each tube will have DNA that all end with the same base but of varying length. 3. Gel electrophoresis is then used to find the comparative size of the bases 4. The smallest fragment will end with the nucleotide of the lane it is in, the second smallest will end with the nucleotide of the lane that it is in and so forth to the largest fragment which ends with the nucleotide of the lane that it is in. 5.
In this way it is possible to sequence up to around 500 bases , restriction mapping can be used to cut up longer sequences and then sequence each fragment and place them together.
Both
DN A
sequencing and restriction mapping tends to be done automatically and analysed by machines.
Genetic screening is used to detect mutant alleles or the development of oncogenes and be used to give lifestyle
advice. 1. The order of bases of the target gene is f irst sequenced and a fragment of DNA with complementary bases produced and radioactively labelled. 2. PCR is used to copy the probe several times and the probe is added to single stranded DNA fragments. If the target gene is present the probe will bind to it. 3. X-ray film can be used to find out whether or not the gene is present as the X-ray film will be exposed by the radioactive probe. Genetic fingerprinting is use to compare the DNA of two individuals and is based on the fact that the DNA of every
individual, barring identical twins , is unique. The technique relies on the non-coding introns in DNA that contain repetitive sequences called core sequences and are different in all individuals, except identical twins, although the more similar two individuals are the more similar their core sequences. 1. DN A is extracted from a sample and often the quantity of DNA is increased by PCR 2. They are then digested by restriction endonucleases to cut close to, but not within, core sequences 3. The fragments are separated using gel electrophoresis and then immersed in alkali to separate the strands 4. DN A fragments are transferred onto a nylon film using a technique known as southern blotting a.
A thin nylon membrane is laid over the gel
b. The membrane draws up liquid containing DN A c.
DNA fragments occupy the same position as on the gel and are fixed on the nylon using UV light
5. Radioactive DNA probes with complementary base sequences are then used to bind with core sequences. The process is carried out with different probes, each of which binds with different core sequences. 6. An X-ray film is placed on the DNA probe and allowed to develop into a series of bars that can be compared. DN A
fingerprints can be visually checked and if there appears to be a match a scanning machine can be used to
calculate the exact length of the DNA fragments and the odds calculated of someone else having the same fragmentation pattern. DNA fingerprinting can be used in forensic science , paternity tests and the genetic diversity of a population.
