3.8.4.3 Genetic fingerprinting

Genetic fingerprinting is a diagnostic tool used widely in forensic science, plant and animal breeding, and medical diagnosis. It is based on the fact that everyones DNA is difference (except identical twins). It relies on the fact that the genome of most eukaryotic organisms contains many repetitive non-coding bases of DNA. DNA bases which are non-coding are known as VNTRs. For everyone the number and length of VNTRs has a unique pattern. The probability of two individuals having the same VNTRs is very low.

Gel electrophoresis
This is used to separate DNA fragments according to their size. The fragments are placed on an agar gel and a voltage is applied across it. The resistance of the gel means that the larger the fragments the fess they move. In this way fragments of different lengths are separated. If fragments are labelled (e.g with radioactive DNA probes) their position in the gel can be determined by placing a sheet of X-ray film over the agar gel for several hours. The radioactivity from each DNA fragment exposes the film and shows where the fragment is situated.


Genetic fingerprinting
This consists of 5 main stages:

• Extraction
• extract the DNA from the sample by separating it from the rest of the cell
• Digestion
• cut the DNA into fragments
• Separation
• separate the fragments by gel electrophoresis under the influence of electrical voltage
• the gel is immersed in alkali to separate the double strands into single strands
• Hybridisation
• DNA probes are used to bind with VNTRs. Different probes for different target DNA sequences
• Development
• X-ray film is put over the nylon membrane

The pattern of bars of each sample is passed through an automated scanning machine which calculates the length of DNA fragments.

Genetic relationships and variability

Genetic fingerprinting can help resolve questions of paternity. It is also useful in determining genetic variability within a population.


Forensic science
Genetic fingerprinting can establish whether a person is likely to have been at the scene of a crime.

Medical diagnosis
Genetic fingerprinting can help diagnose diseases such as Huntington's disease.


Plant and animal breeding
Genetic fingerprinting can be used to prevent undesirable inbreeding during breeding season. It can also identify plants/animals that have a particular allele of a desirable gene.

3.8.4.2 Differences in DNA between individuals of the same species can be exploited for identification and diagnosis of heritable conditions

Often human diseases are the result of a gene mutation. Recombinant DNA technology has enabled us to diagnose and treat many of these genetic disorders. We need to know where a particular DNA sequence is located. To achieve this we use DNA probes and DNA hybridisation.

DNA probes
A DNA probe is a short single stranded length of DA that has a label attached to make it more identifiable. The most common probes are:

• radioactively labelled probes
• made up of nucleotides with the isotope 32p
• identified using an X-ray film that is exposed by radioactivity
• fluorescent labelled probes
• emit light under certain conditions (e.g when the probe has bound to the target DNA sequence)

DNA probes are used to identify particular alleles of genes in the following ways:

• a DNA probe is made that has base sequences that are complementary to part of the base sequence of the DNA that makes up the allele of the gene that we want to find
• the double stranded DNA that is being tested is treated to separate its two strands
• the separated DNA strands are mixed with the probe which binds to the complementary base sequence on one of the strands. This is known as DNA hybridisation.
• The site at which the probe binds can be identified by the radioactivity/fluorescence that the probe emits

DNA hybridisation

This takes pace when a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases. Before this can take place the two DNA strands must be separated. This is achieved by heating the DNA until its double strands separate (denaturation). When cooled the complementary bases on each strand recombine (anneal) with each other to reform the original double strand.


Locating specific alleles
It is possible to locate a specific allele of a gene using DNA probes and DNA hybridisation. E.g to establish whether an individual possesses a mutant allele that causes a particular genetic disorder:

• we must first determine the sequence of nucleotide bases of the mutant allele we are trying to locate. This can be achieved using sequencing techniques/referring to the genetic library
• a fragment of DNA is produced that has a sequence of bases that are complementary to the mutant allele we are trying to locate
• multiple copies of our DNA probe are formed using PCR
• A DNA probe is made by attaching a marker (e.g a fluorescent dye) to the DNA fragment
• DNA from the person suspected of having the mutant allele we want to locate is heated to separate its two strands
• the separated strands are cooled in a mixture containing many of our DNA probes
• if the DNA contains the mutant allele one of our probes is likely to bind to it because the probe has base sequences that are exactly complementary to those on the mutant allele
• the DNA is washed clean of any unattached probes
• the remaining hybridised DNA will now be fluorescently labelled with the dye attached to the probe
• the dye is detected by shining light onto the fragments causing the dye to fluoresce. This can be seen using a special microscope

Genetic screening

If a mutation arises in a dominant allele all individuals will have the genetic disorder. it is important to screen individuals who may be carriers (heterozygous) of a mutant allele. Screening can determine the probabilities of couples having offspring with a genetic disorder. Genetic screening can also be valuable in the detection of oncogenes. Cancers may develop as a result of mutations that prevent the tumour suppressor genes inhibiting cell division.

Another advantage of genetic screening is personalised medicine. This allows doctors to provide health advice based on an individuals genotype. Furthermore, this can improve genetic counselling.

3.6.4.2 Control of blood glucose concentration

Okay so to start we should know a little bit about hormones:

• They are all produced in glands which secrete the hormone directly into the blood (endocrine glands)
• carried in the blood plasma to the cells on which they act (target cells). These cells have specific receptors on their cell-surface membranes that are complementary to a specific hormone
• are effective in very low concentrations but often have widespread and long lasting effects

One mechanism of hormone action is the secondary messenger model. This is used by two hormones involved in the regulation of blood glucose concentration (adrenaline and glucagon).

The mechanism involving adrenaline is as follows:

• adrenaline binds to a transmembrane protein receptor within the cell-surface membrane of a liver cell
• this binding causes the protein to change shape on the inside of the membrane
• this protein shape change leads to the activation of an enzyme called adenyl cyclase. The activated adenyl cyclase converts ATP to cyclic AMP (cAMP)
• The cAMP acts as a second messenger that binds to protein kinase enzyme changing its shape and therefore activating it
• the active protein kinase enzyme catalyses the conversion of glycogen to glucose which moves out of the liver cell (by facilitated diffusion) and into the blood.

The pancreas

The pancreas is a gland that is situated in the upper abdomen (behind the stomach). It produces enzymes for digestion (protease, amylase, and lipase) and hormones for regulating blood glucose concentration (insulin and glucagon). Scattered throughout the cells that produce digestive enzymes are groups of hormone-producing cells known as islets of Langerhans. These include:

• alpha cells (which are larger and produce glucagon)
• beta cells (which are smaller and produce insulin)

The liver

This is located below the diaphragm. It is made up of cells called hepatocytes. The hormones that the pancreas produces (glucagon and insulin) have an effect in the liver. There are three processes regarding blood sugar which take place in the liver that we need to learn:

• Glycogenisis
• This is the conversion of glucose to glycogen
• when blood glucose concentration is higher than normal the liver removes glucose from the blood and converts it into glycogen.
• Glycogenolysis
• This is the break down of glycogen to glucose
• when blood glucose concentration is lower than normal the liver can convert stored glycogen to glucose which diffuses into the blood to restore the normal blood glucose concentration
• Gluconeogenesis
• This is the production of glucose from sources other than carbohydrate (e.g glycerol and amino acids) when the supply of glycogen is exhausted.

The beta cells of the islets of Langerhans in the pancreas have receptors that detect a rise in blood glucose concentration. They respond by secreting the hormone insulin directly into the blood plasma. 

Factors that affect blood glucose concentration include:

• diet - in the form of glucose absorbed following hydrolysis of other carbohydrates (e.g starch/maltose/lactose/sucrose)
• glycogenolysis - the break down of glycogen to glucose
• gluconeogenesis - the break down of sources other than carbohydrates into glucose

Insulin

The beta cells of the islets of Langerhans in the pancreas have receptors that detect the stimulus of a rise in blood glucose concentration and respond by secreting insulin. Glycoprotein receptors on cell-surface membranes bind specifically with insulin molecules bringing about:

• a change in the tertiary structure of the glucose transport carrier proteins which causes them to change shape and open allowing more glucose into the cells by facilitated diffusion allowing more glucose in by facilitated diffusion
• an increase in the number of carrier proteins responsible for the transport of glucose
• at low insulin concentrations the protein from which these channels are made is part of the membrane vesicles. A rise in insulin concentration results in these vesicles fusing with the cell-surface membrane so increasing the number of glucose transport channels
• activation of the enzymes that convert glucose to glycogen and fat (glycogenesis)

As a result blood glucose concentration is lowered by:

• increasing the rate of absorption of glucose into the cells
• increasing the respiratory rate of the cells which uses up more glucose thus increasing their uptake of glucose
• increasing the rate of conversion of glucose into glycogen in the liver and muscle cells
• increasing the rate of conversion of glucose to fat

This subsequent lowering of the glucose in the blood causes the beta cells to reduce their secretion of insulin

Glucagon

Alpha cells of the islets of Langerhans detect a fall in blood glucose concentration and respond by secreting the hormone glucagon. Glucagon...

• attaches to specific protein receptors on the cell surface membrane of liver cells
• activating enzymes that convert glycogen to glucose
• activate enzymes involved in the conversion of amino acids and glycerol into glucose

Adrenaline

At times of excitement/stress adrenaline is produced by the adrenal glands above the kidneys. This raises the blood glucose by:

• attaching to protein receptors on the cell-surface membrane of target cells
• activating enzymes that causes the breakdown of glycogen to glucose in the liver

Insulin and glucagon act antagonistically. This means that the concentration of glucose is not constant but fluctuates around an optimum point.

Diabetes

There are two forms of diabetes:

• Type 1
• due to the body being unable to produce insulin
• Control: it is controlled by injections of insulin. 
• Type 2
• due to glycoprotein receptors on body cells being lost/losing their responsiveness to insulin
• Control: regulate the intake of carbohydrate in the diet and match this to an amount of exercise

3.6.4.3 Control of blood water potential

The homeostatic control of water potential of the blood is called osmoregulation. Mammals have 2 kidneys made up of the following:

• a fibrous capsule - an outer membrane that protects the kidney
• a cortex - a lighter coloured outer region made up of Bowman's capsules convoluted tubules. and blood vessels
• medulla - a darker coloured inner region made up of loops of Henle, collecting ducts, and blood vessels
• renal pelvis - a funnel shaped cavity that collects urine into the ureter
• ureter - a tube that carries urine to the bladder
• renal artery - supplies the kidney with blood from the heart via the aorta
• renal vein - returns blood to the heart via the vena cava

The nephron is the functional unit of the kidney. It is a narrow tube with two twisted ends separated by a central hairpin loop. Each one is made up of a:

• Bowman's capsule - cup shaped and surrounds a mass of blood capillaries known as the glomerulus. The inner layer is made up of podocytes
• proximal convoluted tubule - a series of loops surrounded by blood capillaries. Made of thin epithelial cells which have microvilli
• Loop of Henle - a long hairpin loop that extends from the cortex into the medulla of the kidney and back again surrounded by blood capillaries
• distal convoluted tubule - a series of loops surrounded by blood capillaries. Made up of epithelial cells but is surrounded by fewer capillaries than the proximal convoluted tubule
• collecting duct - a tube which a number of distal convoluted tubules empty into. Lined by epithelial cells and widens as it empties into the pelvis of the kidney.

Blood vessels:

• afferent arteriole arises from the renal artery and supplies the nephron with blood
• glomerulus is a many branched knot of capillaries from which fluid is forced out the blood
• efferent arteriole is a tiny vessel that leaves the glomerulus
• blood capillaries is a concentrated network of capillaries that surrounds the proximal convoluted tubule, the loop of Henle, and the distal convoluted tubule from where they reabsorb mineral salts glucose and water. They merge together to form the renal vein.

The nephron works in 4 stages:

• formation of the glomerular filtrate by ultrafiltration
• the walls of the glomerular capillaries are made up of endothelial cells with pores between them. There is a build up of hydrostatic pressure as the diameter of the afferent arteriole is greater than of the efferent arteriole. As a result water/glucose/mineral ions are squeezed out of the capillary to form the glomerular filtrate. Large proteins/blood cells cannot pass across.
• The inner layer of the renal capsule is made up of highly specialised cells canned podocytes. These have gaps between their branches and the filtrate can pass between these cells.
• the endothelium of the glomerular capillaries have spaces up to 100nm wide

• reabsorption of glucose and water by the proximal convoluted tubule
• in the proximal convoluted tubule almost 85% of the filtrate is reabsorbed back into the blood. Due to ultrafiltration most small molecules are removed but most of these are useful so they get reabsorbed
• the proximal convoluted tubules are adapted to reabsorb substances by having epithelial cells that have:
• microvilli
• infoldings to give a large surface area
• a high density of mitochondria to provide ATP for active transport
• The process is as follows:
• sodium ions are actively transported out of the cells lining the proximal convoluted tubule into blood capillaries which carry them away so the sodium concentration of these cells is lowered
• sodium ions diffuse down a concentration gradient from the lumen into the epithelial lining cells by carrier proteins
• these carrier proteins carry another molecule (glucose/amino acids/chloride ions) with them (co-transport)
• the molecule which have been co-transported into the cells of the proximal convoluted tubule diffuse into the blood

• maintenance of a gradient of sodium ions in the medulla by the loop of Henle
• The loop of Henle is responsible for water being reabsorbed from the collecting duct by concentrating the urine so that it has a lower water potential than the blood. The concentration of urine produced is directly related to the length of the loop of Henle. It has two regions: The descending limb (highly permeable to water), and the ascending limb (impermeable to water)
• The loop of Henle acts as a countercurrent multiplier:
• sodium ions are actively transported out of the ascending limb of the loop of Henle using ATP provided by mitochondria in the cells of its wall
• This creates a low water potential/high ion concentration in the region of the medulla between the two limbs (the intestinal region). The thick walls are impermeable to water so very little escapes the ascending limb.
• The walls of the descending limb are permeable to water so it passes out by osmosis into the interstitial space and enters blood capillaries
• the filtrate progressively loses water as it moves down the descending limb lowering its water potential
• at the base sodium ions diffuse out the filtrate and as it moves up the ascending limb these ions are also actively pumped out so the filtrate develops a progressively high water potential
• in the space between the ascending limb and the collecting duct there is a gradient of water potential with the highest in the cortex sand the lowest in the the further down into the medulla
• the collecting duct is permeable to water so as the filtrate moves down it water passes out by osmosis. This passes into blood vessels that occupy this space and is carried away
• As water passes out of the filtrate its water potential is lowered but the water potential is also lowered in the interstitial space so water continues to move out by osmosis down the whole length of the collecting duct. The countercurrent multiplier ensures that there is always a water potential gradient drawing water out of the tubule
• reabsorption of water by the distal convoluted tubule and collecting ducts
• the main role of the distal convoluted tubule is to make final adjustments to the water and salts that are reabsorbed and to control pH of the blood by selecting which ions to reabsorb. The homeostatic control of osmoregulation in the blood is achieved by a hormone that acts on the distal convoluted tubule and the collecting duct.

The body responds to a fall in water potential of the blood as follows:

• osmoreceptors in the hypothalamus (in the brain) detect a fall in water potential and water is lost from the osmoreceptor cells which causes the cell to shrink causing the hypothalamus to produce ADH (antidiuretic hormone)
• ADH passes into the pituitary gland from where it is secreted into the capillaries
• ADH passes into the blood to the kidney
• protein receptors on the cell surface membrane of these cells bind to ADH leading to the activation of an enzyme (phosphorylase). This causes vesicles within the cell to move to and fuse with the cell surface membrane
• These vesicles contain pieces of plasma membrane that have numerous water channel proteins (aquaporins) and the number of water channels is significantly increased when they fuse
• this increases the permeability of the collecting duct to urea which passes out lowering the water potential of the interstitial space
• this causes more water to leave the collecting duct by osmosis and reenter the blood
• this prevents the water potential of the blood from getting any lower

3.6.3 Skeletal muscles are stimulated to contract by nerves and act as effectors

Muscles are effector organs that respond to nervous stimulation by contracting and so bring about movement. They act in antagonistic pairs against an incompressible skeleton. There are three types:

• cardiac muscle (heart)
• smooth muscle (walls on blood vessels and the gut)
• skeletal muscle (attached to bone, under voluntary control)

Individual muscles are made up of tine fibres called myofibrils. They produce very little force but all together are extremely powerful. They are lined parallel to maximise force. Individual muscle cells do not have nuclei etc. The cells have become fused together into muscle fibres which share nuclei and cytoplasm (known as sarcoplasm). Within the sarcoplasm is a large concentration of mitochondria and endoplasmic reticulum.

Myofibrils are made up of two types of protein filament:

• actin
• this is thinner and consists of two strands twisted around eachother
• tropomyosin forms a fibrous strand around the actin filament
• myosin
• this is thicker and consists of long rod-shaped tails with bulbous heads that project to the side

Myofibrils appear striped due to alternating dark and light bands. Light bands are isotropic bands (I-bands) - here the thick (myosin) and thin (actin) filaments do not overlap hence they are lighter in colour. Dark bands are called anisotropic bands (A-bands) - here the myosin and actin filaments overlap hence it appears darker here. At the centre of each A-band is a lighter region known as the H-zone and at the centre of each I-band is a line called the Z-line. The sarcomere is the distance between adjacent Z-lines. When the muscle contracts the sarcomeres shorten.

There are two types of muscle fibre:

• slow-twitch fibres
• contract more slowly and provide less powerful contractions but over a long period
• adapted to endurance work (e.g running a marathon)
• they are suited to this role by being adapted to aerobic respiration to avoid a build up of lactic acid (this would cause them to function less effectively and prevent long-duration contraction)
• they have a large store of myoglobin, a rich supply of blood vessels (to deliver o2 and glucose for aerobic respiration), and numerous mitochondria to produce ATP
• fast-twitch fibres
• contract more rapidly producing more powerful contractions but only for a short period of time
• adapted to intense exercise so are more common in muscles that need to do short bursts of intense activity
• they have thicker and more numerous myosin filaments, a high concentration of glycogen, a high concentration of enzymes involved in anaerobic respiration which provide ATP rapidly, a store of phosphocreatine (a molecule that can rapidly generate ATP from ADP in anaerobic conditions)

A neuromuscular junction is the point where a motor neurone meets a skeletal muscle fibre. It is covered in 3.6.2.2.


The contraction of a skeletal muscle will move a part of the skeleton (e.g a limb) in one direction but the same muscle cannot move it in the other direction as muscles can only pull (not push). A second muscle working antagonistically is required to move the limb in the opposite direction.

The process of contraction involves the actin and myosin filaments sliding past one another so it is known as the sliding filament mechanism. This is supported by the changes seen in band pattern on myofibrils. When a muscle contracts...

• the I-band becomes narrower
• the Z-lines move closer together (the sarcomere shortens)
• the H-zone becomes narrower
• The A-band remains the same width as the width of this band is determined by the length of the myosin filaments and they do not get shorter.

Here's how the mechanism works:

• an action potential reaches many neuromuscular junctions simultaneously causing calcium ion protein channels to open and calcium ions to diffuse into the synaptic know
• the calcium ions cause the synaptic vesicles to fuse with the presynaptic membrane and release their acetylcholine into the synaptic cleft
• acetylcholine diffuses across the synaptic cleft and binds with receptors on the muscle cell surface membrane causing it to depolarise
• the action potential travels into the fibre through a system fo T-tubules that are extensions of the cell surface membrane and branch throughout the cytoplasm of the muscle (the sarcoplasm)
• the tubules are in contact with the endoplasmic reticulum of the muscle which has actively transported calcium ions from the cytoplasm of the muscle leading to a low concentration of calcium ions in the cytoplasm
• the action potential opens the calcium ion protein channels on the endoplasmic reticulum and calcium ions diffuse into the muscle cytoplasm down a concentration gradient
• the calcium ions cause the tropomyosin molecules the were blocking the actin binding sites to pull away
• ADP molecules attach to the myosin heads so now they are in a state to bind to actin filaments forming cross-bridges
• once attached to the actin filament the myosin heads change their angle pulling the actin filament along as they do so and releasing a molecule of ADP
• an ATP molecule attaches to each myosin head causing it to become detached from the actin filament
• the calcium ions activate the enzyme ATPase which hydrolyses ATP to ADP. This provides the energy for the myosin head to return to its original position
• the myosin head with an attached ADP molecule then reattaches itself further along the actin filament and the cycle is repeated as long as the concentration of calcium ions in the myofibril remains high
• when nervous stimulation ceases the calcium ions are actively transported back into the endoplasmic reticulum using energy from the hydrolysis of ATP
• the reabsorption of the calcium ions allows tropomyosin to block the actin filament binding sits
• myosin heads are now unable to bind to actin filaments and contraction ceases

So this all requires A LOT of energy. This is supplied by the hydrolysis of ATP to ADP + Pi. This energy is needed for the movement of myosin heads and the reabsorption of calcium ions into the endoplasmic reticulum by active transport. In an active muscle there is a great demand for ATP. Most energy is regenerated from ADP during the respiration of glucose in the mitochondria but this requires oxygen. In very active muscles we can rapidly generate ATP anaerobically using phosphocreatine

3.6.2.1 Nerve impulses

Neurones are specialised cells adapted to carry electrochemical changes (nerve impulses) from one part of the body to another. A myelinated motor neurone is made up of:

• a cell body containing the usual organelles including a large amount of RER which is associated with the production of proteins and neurotransmitters
• dendrons which are extensions of the cell body which subdivide into dendrites that carry nerve impulses towards the cell body
• an axon which is a single long fibre that carries nerve impulses away from the cell body
• Schwann cells which surround the axon protecting it providing electrical insulation. They also carry out phagocytosis
• A myelin sheath which forms a covering to the axon and is made up of the membranes of the Schwann cells
• nodes of Ranvier are constrictions between adjacent Schwann cells where there is no myelin sheath

Resting potential

The inside of an axon is negatively charged relative to the outside. This is a resting potential and in this condition the axon is said to be polarised and this can be established due to the following events:

• sodium ions are actively transported out of the axon by the sodium-potassium pump
• potassium ions are actively transported into the axon by the sodium-potassium pump
• the active transport of sodium ions is greater than the active transport of potassium ions so an electrochemical gradient is established
• the sodium ions begin to diffuse back in while the potassium ions begin to diffuse back out
• most of the gates in the channels that allow potassium through are open whilst most of the gates in the channels that allow sodium ions to move through are closed

When a stimulus is detected by a receptor it causes a temporary reversal of the charges either side of the axon. If the stimulus is large enough the inside of the membrane becomes a positive charge. This is known as an action potential and the axon membrane is now said to be depolarised. This occurs because the voltage-gated channels change shape opening/closing depending on the voltage across the membrane:

1. at resting potential some potassium voltage-gated channels are open (the permanently open ones) but the sodium voltage-gated channels are closed
2. the energy of the stimulus causes some sodium voltage-gates channels to open causing sodium ions to diffuse into the axon through these channels along an electrochemical gradient. This triggers a reversal in the potential difference across the membrane as they are positively charged
3. as sodium move in more sodium channels open causing a greater influx (this is positive feedback)
4. once an action potential of ~40mV is established the voltage gates on the sodium channels close to prevent further influx of sodium ions and the voltage gates in potassium channels begin to open
5. lots of potassium ions diffuse out causing more potassium ions to diffuse out starting repolarisation of the axon
6. the outward diffusion of the potassium ions causes a temporary overshoot of electrical gradient with the axon inside being more negative relative to the outside than usual (hyperpolarisation). The potassium ion channels close and the sodium potassium pump causes sodium to move out and potassium to move in reestablishing the resting potential. The axon is said to be repolarised.

Once created the action potential moves rapidly along the axon. As one region of the axon produces an action potential and depolarises it acts as a stimulus for the depolarisation of the next region of the axon. The action potential is a wave of depolarisation and the previous region of the membrane returns to its resting potential by undergoing repolarisation.

How an action potential moves along an unmyelinated axon:

• at resting potential the axon membrane is polarised and negatively charged on the inside relative to the outside
• a stimulus causes an influx of sodium ions and hence a reversal of charge on the axon membrane. This is the action potential and the membrane is depolarised
• the localised electric currents established by the influx of sodium ions cause the opening of sodium voltage-gated channels a little further along resulting in depolarisation in that region. Behind this region sodium channels close and potassium channels open beginning repolarisation
• the action potential is propagated in the same way further along the axon
• repolarisation allows sodium ions to be actively transported out and potassium ions to be actively transported in returning the axon to its resting potential

How an action potential moves along a myelinated sheath: In myelinated axons the sheath of myelin acts as an electrical insulator preventing action potentials from forming. At intervals there are breaks in the insulation (nodes of Ranvier). Action potentials can occur at these points and so the action potential effectively jumps from node to node in a process known as saltatory conduction. An action potential travels along a myelinated  neurone faster than along the axon of an unmyelinated one of the same diameter.

The transmission of an action potential along the axon of a neurone is the nerve impulse. A number of factors affect the speed at which an action potential passes along an axon:

• the myelin sheath
• the diameter of an axon - the larger the diameter the faster the speed
• temperature - this affects the rate of diffusion of ions so a higher temperature means a faster nerve impulse

Once an action potential has been created in any region of an axon there is a period after where inward movement of sodium ions is prevented as the sodium voltage-gated channels are closed. During this time it is not possible for a further action potential to be generated. This is known as the refractory period. It has 3 purposes:

• ensures action potentials are propagated in one direction only as action potentials cannot be propagated in a region of refractory
• produces discrete impulses as a new action potential cannot be formed immediately behind the first one ensuring action potentials are separated from one another
• it limits the number of action potentials as they are separated from one another

3.6.1.1 Survival and response

A stimulus is a detectable change in the internal/external environment of an organism that leads to a response in the organism. This increases the organisms chance of survival (e.g the ability to detect and move away from prey, or detect and move towards a food source).

So we need to know quite a bit about plants (in particular, flowering plants). Plants have no nervous system so their responses to external stimuli involve hormone-like substances known as plant growth factors. They are produced in small quantities and an example is IAA which is an auxin. IAA controls plant elongation. Growth factors can move from growing regions to other tissues (meaning that they are not confined to the tissue they were produced in) and bring about a change in growth in the following way:

1. Cells in the shoot tip produce IAA
2. the IAA is transported down the shoot evenly throughout all regions
3. Light causes the movement of IAA from the light side to the shaded side of the shoot so a greater concentration of IAA builds up on the shaded side
4. The cells on the shaded side elongate more as there is a greater concentration of IAA here
5. The shaded side elongates faster than the light side which causes the shoot tip to bend towards the light

IAA inhibits root growth:

1. Cells in the root tip produce IAA
2. the IAA is transported initially to all sides of the root
3. Gravity influences the movement of IAA from the upper side to the lower side
4. A greater concentration builds up on the lower side meaning the cells here elongate less than those on the upper side
5. This causes the root to bend down towards gravity.

IAA has a number of effects on plant cells including increasing the plasticity of their cell walls. This response only occurs in young cell walls where cells are able to elongate. As cells mature they develop greater rigidity meaning that older parts of the shoot/root will not respond. The acid growth hypothesis is the current way in which we think IAA increases the plasticity of cells. It involves the active transport of hydrogen ions from the cytoplasm into the cell walls causing the cell wall to become more plastic allowing the cell to elongate.

A taxis is a simple response whose direction is determined by the direction of the stimulus. As a result a motile organism responds directly to the environmental changes by moving away from an unfavourable one. Taxis are classified according to whether the movement is towards (positive) or away from (negative taxis) the stimulus and by the nature of the stimulus. Some examples are as follows:

• Positive phototaxis - single celled algae move towards light to increase their chances of survival as they are photosynthetic
• Positive chemotaxis - some bacteria move towards a region of high glucose concentration. This increases their chance of survival because they use glucose as a food source.

A kinesis is a form of response in which the organism does not move towards or away from a stimulus. Instead it changes its speed and also the rate it changes direction. If it crosses a line between favourable and unfavourable conditions its rate of turning increases which raises its chances of quickly returning to a favourable environment. If it has moved a considerable distance in an unfavourable environment its rate of turning decreases so it moved in a long straight line before sharply turning as this is likely to bring the organism into a new region with favourable conditions.

A tropism is the growth of part of a plant in response to a directional stimulus. The type of response is named after the stimulus and the direction the organism moves relative to it. Examples include:

• positive phototropism - plant shoots grow towards the light so their leaves are in a more favourable position for photsynthesis
• Plant roots grow away towards gravity (positive gravitropism). This increases their probability to find water and mineral ions.

Stimuli are detected by receptors which are specific to one type of stimulus. A coordinator formulates a suitable response, which is produced by an effector. Animals also have a nervous system in addition to hormonal communication. This has many different receptors and effectors which are linked to a central coordinator.

The spinal cord is a column of nervous tissue running along the back which lies inside the vertebral column for protection. Pairs of nerves emerge at intervals along the spinal cord. A reflex is rapid, short-lived, and localised. The pathway of neurones involved is known as a reflex arc:

1. stimulus 
2. receptor
3. sensory neurone
4. relay neurone/coordinator
5. motor neurone
6. effector
7. response

Reflex actions are important as they make survival more likely. They are important for the following reasons:

• involuntary so do not require the decision powers of the brain so the brain is free to carry out more complex responses
• they protect the body from harm and are effective from birth so do not have to be learnt
• they are fast as the neurone pathway is short with very few synapses
• the absence of the decision making process also makes it fast

3.6.4.1 Principles of homeostasis and negative feedback

Homeostasis in mammals involves physiological control systems that maintain the internal environment within restricted limits. It is very important for many reasons, including:

• enzymes (e.g those involved in biochemical reactions within cells/other proteins such as channel proteins are sensitive to changes in pH and temperature). Any changes from the optimum reduce the rate of reaction and, if severe enough, may denature the enzyme and the reaction will cease altogether. Maintaining a fairly constant environment means that reactions take place at a suitable rate.
• Changes to the water potential of blood/tissue fluid may cause cells to shrink/expand as a result of water leaving/entering via osmosis. This means the cells cannot operate normally.
• The maintenance of a constant blood glucose concentration is essential to ensure a constant water potential. A constant blood glucose concentration also ensures a reliable source of glucose for respiration by cells.

Positive feedback

This occurs when a deviation from an optimum causes changes that result in an even greater deviation from the normal. As example occurs in neurones where a stimulus leads to a small influx of sodium ions (this increases the permeability of the neurone membrane to sodium ions and more ions enter).


Negative feedback
Negative feedback is when the change produced by the control system leads to a change in the stimulus detected y the receptor and turns the system off. This restores systems to their original level. 


Control of any system involves a series of stages:

• the optimum point
• a receptor
• a coordinator
• an effector
• a feedback mechanism

The possession of separate mechanisms involving negative feedback controls departures in different directions from the original state, giving a greater degree of homeostatic control as the return to the optimum can be brought about faster.

For example, if there is a fall in blood glucose concentration this is detected by receptors on the cell-surface membrane of alpha cells in the pancreas. These secrete glucagon which causes liver cells to convert glycogen to glucose to raise the blood glucose concentration. There is now reduced stimulus so the secretion of glucagon reduces.

If blood glucose concentration rises insulin will be produced from beta cells in the pancreas. Insulin increases the uptake of glucose by cells (it is converted to glycogen and fat). There is now reduced stimulus so the production of insulin reduces.

3.8.3 Using genome projects

The Human Genome Project (HGP) was an international scientific research project with the goal of determining the sequence of nucleotide base pairs that make up human DNA, and of identifying and mapping all of the genes of the human genome.Projects to determine the entire DNA nucleotide base sequence of organisms work by mapping the DNA base sequences that make up the genes of the organism and then map these genes on the individual chromosome of that organism. In this way a complete map of all the genetic material in an organism (the genome) is obtained.

The HGP would have been impossible without the use of bioinformatics (the science of collecting and analysing complex biological data, e.g genetic codes). It uses computers to read, store, and organise biological data at a very fast rate and utilises algorithms to analyse and interpret biological data.

Determining the complete DNA base sequence of an organism uses the technique of whole-genome shotgun (WGS) sequencing. This involves researchers cutting the DNA into many small easily sequenced sections and then using computer algorithms to align overlapping segments to assemble the entire genome. Sequencing methods are continuously updated. This and increased automation of the processes involved have lead to extremely rapid sequencing of whole genomes.

One outcome of the HGP is that lots of medical advances have been made. E.g over 1.4 million SNPs (single nucleotide polymorphisms) have been found in the human genome. SNPs are single-base variations in the genome that are associated with disease/disorders. Medical screening of individuals has allowed quick identification of potential medical problems (this is good for early intervention).

The proteins a genome codes for are known as the proteome (all the proteins produced by the genome). A protein is only produced when a gene is switched on. The cellular proteome is all the proteins produced in a given type of cell at a given time under specific conditions. The complete proteome is all the proteins produced in a given organism at a given time under specific conditions.

Determining the genome and proteome of simpler organisms
We sequence the genomes of prokaryotic/single-celled eukaryotic cells to gain information to help cure disease and provide knowledge of genes that can be usefully exploited. E.g ones from organisms that can withstand extreme/toxic environmental conditions and so have potential uses in cleaning up pollutants or in manufacturing biofuels. Determining the proteome of prokaryotes is relatively easy because:

• the vast majority of prokaryotes have just one circular piece of DNA that is not histone associated
• there are none of the non-coding portions (introns) of DNA which are typical of eukaryotic cells

Knowledge of the proteome of prokaryotes has a number of applications. One is the identification of the proteins that act as antigens on the surfaces of human pathogens. These antigens can be used in vaccines against diseases caused by these pathogens. The antigens can be manufactured and then administered to people in appropriate doses. In response, memory cells are formed etc.

Determining the genome and proteome of complex organisms

The problem in complex organisms is translating knowledge of the genome into the proteome. This is because the genome of complex organisms contains many introns (non-coding genes) as well as others that have a role in regulating other genes.

3.8.2.3 Gene expression and cancer

Cancer is a group of diseases caused by damage to the genes that regulate mitosis and the cell cycle. This leads to unrestrained growth of cells and as a consequence an abnormal group of cells (a tumour) develops and constantly expands in size.

It is important to realise that not all types of tumour are cancerous. Cancerous tumours are known as malignant tumours, non-cancerous tumours are known as benign tumours). We need to know the main characteristics of them:

Benign (non-cancerous)	Malignant (cancerous)
Grow to a large size very slowly	Can also grow to a large size, but very rapidly
The cell nucleus has a normal appearance	The cell nucleus is often larger and darker due to an abundance of DNA
Cells are often specialised (differentiated)	Cells become unspecialised (de-differentiated)
Cells produce adhesion molecules that make them stuck together so they remain within the tissue from which they arise (primary tumours)	Cells do not produce adhesion molecules and so they tend to spread to other regions of the body (metastasis) (secondary tumours)
Tumours are surrounded by a capsule of dense tissue so remain as a compact structure	Tumours are not surrounded by a capsule and can therefore grow finger-like projections into surrounding tissue
Much less likely to be life threatening but can potentially disrupt the functioning of a vital organ	More likely to be life threatening as abnormal tumour tissue replaces normal tissue
Tend to have localised effects on the body	Often have systemic effects such as weight loss and fatigue
Can usually be removed by surgery	Removal usually involves radio/chemotherapy and also surgery
Rarely reoccur after treatment	More frequently reoccur after treatment

DNA analysis of tumours has shown that cancer cells are derived from a single mutant cell. Further mutation in one of the descendent cells leads to other changes that cause subsequent cells to be different from normal cells in growth and appearance. The two main types of genes that play a role in cancer are tumour suppressor genes and oncogenes. An importance difference between oncogenes and tumour suppressor genes is that while oncogenes cause cancer as a result of the activation of proto-oncogenes, tumour suppressor genes cause cancer when they are inactivated.

Oncogenes
Most oncogenes are mutations from proto-oncogenes. Proto-oncogenes stimulate a cell to divide when growth factors attach to a protein receptor on its cell-surface membrane. This activates the genes that cause the DNA to replicate and the cell to divide. If a proto-oncogene mutates into an oncogene it can become permanently activated (switched on) for two reasons:

• The receptor protein on the cell-surface membrane can be permanently activated so that cell division is switched on even in the absence of growth factors
• The oncogene may code for a growth factor that is then produced in excessive amounts stimulating excessive cell division

The result is that cells divide too rapidly and out of control resulting in either a tumour or a cancer. A few types of cancer are caused by inherited mutations of proto-oncogenes that cause the oncogene to be activated. Most cancer-causing mutations involving oncogenes are acquired not inherited.

Tumour supressor genes
Tumour suppressor genes slow down cell division (they suppress tumours/cell division), repair mistakes in DNA, and tell cells when to die (apoptosis). They have the opposite role to proto-oncogenes. A normal tumour suppressor gene maintains normal rates of cell division which prevents tumours. If a tumour suppressor gene mutates it becomes inactive/switched off. As a result the inhibition of cell division stops and cells begin to grow out of control. The mutated cells are structurally and functionally different from normal cells. Many die but those that survive can make clones of themselves and form tumours. Some cancers are caused by inherited mutations of tumour suppressor genes but most are acquired. 

Abnormal methylation of tumour suppressor genes
Abnormal DNA methylation is common in the development of a variety of tumours. The most common abnormality is hypermethylation/increased methylation. The process whereby this leads to cancer is as follows:

• Hypermethylation occurs in a specific region (a promoter region) of tumour suppressor genes
• This leads to the tumour suppressor gene being inactivated
• As a result transcription of the promoter regions of tumour suppressor genes is inhibited
• As the tumour suppressor gene normally slows the rate of cell division it's inactivation leads to increased cell division and the formation of a tumour.

Furthermore, hypomethylation/decreased methylation can occur in oncogenes where is leads it leads to their activation hence formation of tumours.

Oestrogen and breast cancer

Oestrogen plays an important role in regulating the menstrual cycle in women. The fat cells of breasts tend to produce more oestrogen after menopause. These locally [produced oestrogens appear to trigger breast cancer in postmenopausal women. Once a tumour has developed it further increases oestrogen concentration which therefore leads to increased tumour development. White blood cells that are drawn to the tumour also increase oestrogen production. Basically how this works is, if oestrogen binds to a gene that controls cell division and growth then the gene will be activated and its continued division could produce a tumour.  It is also known that oestrogen causes proto-oncogenes to develop into oncogenes which leads to the development of a tumour.

3.8.2.2 Regulation of transcription and translation

In eukaryotes, transcription of target genes can be stimulated or inhibited when specific transcriptional factors move from the cytoplasm into the nucleus. The general principles involved in controlling gene expression by controlling transcription are as follows:

• For transcription to begin the gene must be switched on by specific molecules (transcriptional factors) that move from the cytoplasm into the nucleus
• Each transcriptional factor has a site that binds to a specific bas sequence of the DNA in the nucleus
• When it binds it causes this region of DNA to begin the process of transcription
• mRNA is produced and the information it carries is translated into a polypeptide (this is translation)
• When a gene is not being expressed (it is switched off), the site on the transcriptional factor that binds to DNA is not active
• As the site is inactive it cannot cause transcription and polypeptide synthesis.

So we need to know a bit about the role of the steroid hormone oestrogen in initiating transcription. Well, hormones like oestrogen can switch on a gene and start transcription by combining with a receptor site on the transcriptional factor. This then activates the DNA binding site on the transcriptional factor (by causing it to change shape):

• Oestrogen, a lipid-soluble molecule, diffuses easily through the phospholipid bilayer of cell-surface membranes
• Once inside the cytoplasm oestrogen binds with a site on a receptor molecule of the transcriptional factor. They are complimentary to one another
• By binding with the site the oestrogen changes the shape of the DNA binding site on the transcriptional factor, activating it
• It can now bind to DNA
• The transcriptional factor enters the nucleus (through a nuclear pore) and binds to a specific base sequence of DNA
• This stimulates transcription of the gene that makes up that portion of DNA

Whilst genes determine the features of an organism, the environment can influence the expression of these genes. It is now believed that environmental factors can cause heritable changes in gene function without changing the base sequence of DNA. This process is known as epigenetics. This provides explanations as to how environmental influences such as diets, stress, toxins (etc), can alter the genetic inheritance of an organism's offspring. So, now about how it works. Basically, we already know that DNA is wrapped around proteins called histones. We now know that both the DNA and histones are covered in chemicals (tags). These chemicals/tags form the epigenome. The epigenome determines the shape of the DNA-histone complex. E.g it keeps genes that are inactive tightly packed in arrangement ensuring they cannot be read (epigenetic silencing). It can also unwrap genes so the DNA is exposed and can be easily transcribed (switching on these particular genes). Unlike DNA, the epigenome is not fixed (it is flexible). It is flexible because its chemical tags respond to environmental changes, factors such as stress and diet can cause the chemical tags to adjust the wrapping/unwrapping, switching genes on/off.

The epigenome of a cell is an accumulation of the signals it has received during its lifetime. It acts a bit like a cellular memory. In early development the signals come from within the cells of the foetus. The nutrition provided by the mother is important in shaping the epigenome at this stage (this is why it is imperative that pregnant woman keep a good diet and don't smoke etc).After birth environmental factors affect the epigenome (although signals, such as hormones, from within the body can still influence it). These factors cause the epigenome to activate/inhibit a specific set of genes. The environmental signal stimulates proteins to carry its message inside the cell from where it is passed by a series of other proteins into the nucleus. Here the message passes to a specific protein which can be attached to a specific sequence of bases on the DNA. Once attached the protein can change:

• acetylation of histones, leading to the activation/inhibition of a gene
• methylation of DNA by attractive enzymes that can add/remove methyl groups.

Okaaay so what does any of that actually mean. Well, when the association of histones with DNA is weak the DNA-histone complex is less condensed meaning that the DNA is accessible by transcriptional factors which can initiate transcription (basically, the gene is switched on). When the association is stronger, the reverse occurs and the gene is switched off. Condensation of the DNA histone complex inhibits transcription. It can be brought about by decreased acetylation of the histones or by methylation of DNA. So how do these processes work?

Decreased acetylation of associated histones
Acetylation is the process whereby an acetyl group is transferred to a molecule. In this case, acetylcoenzyme A donates an acetyl group. Deacetylation is the removal of an acetyl group from a molecule. Decreased acetylation increase the positive charges on histones as acetyl groups are negatively charged. This increases the attraction of the histones to the phosphate groups of DNA. The association between DNA and histones becomes stronger and the genes are switched off.

Increased methylation of DNA
Methylation is the addition of a methyl group to a molecule. In this case a methyl group is added to the cytosine bases of the DNA. It inhibits transcription in the following ways:

• prevents the binding of transcriptional factors to DNA
• attracts proteins that condense the DNA-histone complex by inducing the deacetylation of histones.

Epigenetic changes can be responsible for certain diseases. Altering the epigenetic process can cause abnormal activation/silencing of a gene. In some cases the activation of a normally inactive gene can cause cancer. In other cases, the inactivation (silencing) of a usually active gene causes a disease.

In specific sections of DNA (near promoter regions) that have no methylation in normal cells. In cancer cells these regions become highly methylated causing genes that should be active to switch off.

Whilst epigenetics do not alter the sequence of bases in a DNA molecule they can increase the incidence of mutations. For example, some active genes help to repair DNA (preventing cancers). Individuals with various types of inherited cancer have increased methylation of these genes causing the gene to be switched off. As a result, the damage to base sequences in DNA are not repaired. This can lead to the development of cancer.

It's not all bad news though, we can also use epigenetic treatments to counteract the epigenetic changes that cause certain genes to be activated/silenced. The treatments use drugs to inhibit certain enzymes involved in either histone acetylation or DNA methylation. E.g. drugs that inhibit enzymes that cause DNA methylation can reactivate silenced genes. 

Epigenetics have also been used in diagnostics tests to detect the early stages of diseases such as cancer/brain disorders/arthritis. The tests can identify the level of DNA methylation and histone acetylation at an early stage of disease. This allows patients to seek treatment asap.

RNA interference
This is the last little bit in this section. Basically, in eukaryotes (and some prokaryotes!) the translation of mRNA produced by a gene can be inhibited by breaking mRNA down before its coded information can be translated into a polypeptide.One type of RNA molecule that may be involved is small interfering RNA (siRNA). This mechanism involved small double-stranded sections of siRNA and operates as follows:

• An enzyme cuts large double stranded RNA molecules into smaller sections known as siRNA
• One of the two siRNA strands combines with an enzyme
• The siRNA molecule guides the enzyme to a mRNA molecule by pairing up its bases with the complimentary ones on a section of the mRNA molecule
• Once in position the enzyme cuts the mRNA into smaller sections
• the mRNA is no longer capable of being translated into a polypeptide meaning that the gene has not beed expressed (so it has been silenced/blocked)