Biology A2 Unit 5

Revision

Teacher 1 – Control in Organisms 3.5.1 3.5.5

Survival and Response

Organisms increase their chance of survival by responding to their environment. Their behaviour is most likely going to be in search of food or shelter from predators.

Tropisms responses to unidirectional stimuli. Movement toward or away from the stimulus such as gravitropism/phototropism.

e.g. When a seed germinates the root go down as they are positively gravitropic, whereas the shoot goes upwards and is negatively gravitropic

Taxes and kinesis are simple responses that can maintain a mobile organism in a favourable environment such as one with plentiful food supplies or no predators.

Taxes movement along a gradient of intensity such as temperature:Towards the high intensity (positive taxis)Away from the high intensity (negative taxis)e.g. Daphnia move towards greater light intensity as the phytoplankton

they feed on are found closer to the waters surface where they photosynthesise.

Kinesis change in the rate of movement (not the direction) in response to a stimulus change.

e.g. Woodlice move faster when they are exposed to high light intensity to increase their chance of finding shade again and not losing water as quickly.

Auxin (IAA/Indoleacetic acid)

Auxins act by binding to a specific site on the plasma membrane of a cell activating a H+ pump. The cell wall therefore becomes acidified which activates enzymes. These enzymes then weaken the cell wall which results in an increase in internal pressure; the cell becomes elongated/ GROWS.

The movement of a plant stem towards light is a result of auxin being redistributed (diffused) to the shaded side of the shoot. The cells on this side then grow more than the other side causing a curvature towards the light (positive phototropism).

Movement of auxin

Auxins effect on roots

Auxin has an opposite effect on roots. It is a low concentration of auxin which causes increased growth rate in roots. In both root and shoot the auxin is redistributed (diffused) to the lowers side.

The auxin causes increased growth in the lower side of the shoot causing upwards curvature (negative gravitropism).

The auxin causes decreased growth in the lower side of the root causing downwards curvature (positive gravitropism).

THE NERVOUS SYSTEMNervous System

Central Nervous System (CNS)

Brain Spinal Cord

Peripheral Nervous System

(PNS) Nerves

Autonomic Nervous System

Sympathetic Nervous System

Parasympathetic Nervous System

Somatic Nervous System

Nerve TypesCheck Point - Humans have three types of neurones:

•Sensory neurones have long axons and carry nerve impulses from sensory receptors to the central nervous system.

•Motor neurones also have long axons and carry nerve impulses from the central nervous system to effectors such as muscles.

•Inter-neurones/Relay neurones are usually much smaller cells, with many interconnections. Within the central nervous system they carry impulses between the motor and sensory neurones.

A reflex arc

Structure of a myelinated motor neurone

The myelin sheath

The myelin sheath consists of other nerve cells and allows the nerve impulse to travel quicker.

In the CNS, In the PNS, Oligodendrocytes. Schwann cells.

Resting potentialAn electrical potential difference is

maintained between the outside and inside of the neurone. The inside of the axon is 70mV more negative than the outside. (The membrane is polarised)

This is maintained by:o Large anions such as negatively

charged proteins inside the axono Passive diffusion of Na+ and K+

ions across the membraneo Active transport of the Na+ and

K+ ions by a sodium-potassium pump found in the membrane of the neurone.

A Nerve Impulse

1. Nerve impulse reaches the neurone.2. ‘Voltage-activated’ sodium channels open.3. There is an influx of Na+ ions – when the threshold value is

met - which causes the inside of the neurone to become positively charged (compared with the outside). DEPOLARISATION

4. The action potential continues down the axon.5. The gated sodium channels close and the potassium

channels open.6. The efflux of K+ causes REPOLARISATION of the membrane.

An action potential is an ALL OR NOTHING RESPONSE, unless the threshold value is reached there will be no action potential.

Why does the action potential only travel in one direction?

• The period following the initiation of an action potential when another action potential cannot be generated is called the ABSOLUTE REFRACTORY PERIOD.This absolute refractory period includes:- depolarisation- repolarisation because the sodium gated channels are inactivated when they are closed.

• It is also difficult to initiate another action potential during hyperpolarisation (this is where an excess of K+ ions leave the neurone during repolarisation and the membrane potential is lower than resting potential) but as its not impossible this period is called the RELATIVE REFRACTORY PERIOD.

Speed of TransmissionThere are several factors which affect speed of a nerve impulse:o Temperature Higher temperature = faster transmission, until enzymes and

transport proteins begin to denature.o Diameter of neurone Larger diameter = faster transmission.o Myelination myelinated neurones conduct nerve impulses faster than non-

myelinated neurones because the action potential ‘jumps’ between over the myelin sheath and only needs to be conducted at the nodes of Ranvier.

The transmission of an action potential in a myelinated neurone is called SALTATORY PROPAGATION.

SynapseA nerve impulse can only travel in one

direction at the synapse because:o Only the pre-synaptic neurone has

neurotransmitter-containing vesicles.

o Only the post-synaptic membrane has receptors for the neurotransmitter.

o There is a diffusion gradient of neurotransmitter between the two neurones (across the synaptic cleft).

The secretion of neurotransmitters directly onto the target cell results in a rapid, short-lived and localised response.

At an excitatory synapse...

1. The action potential arrives at the pre-synaptic membrane.2. Calcium channels open and Ca2+ ions enter the neurone.3. This causes the secretary vesicles to move towards and fuse with the

membrane and the neurotransmitter, acetylcholine is released into the synaptic cleft.

4. Acetylcholine diffuses across the Cholinergic Synapse and binds to receptors on the post-synaptic membrane.

5. This causes sodium-ion channels in the post-synaptic neurone to open leading to the action potential being continued down the next neurone.

6. The neurotransmitter is then hydrolysed by the enzyme acetylcholinesterase found in the synaptic cleft and the products are reabsorbed into the pre-synaptic neurone.

7. Acetylcholine is the resynthesised.

When the inhibitory neurotransmitter binds to the post-synaptic membrane, K+ and Cl- channels open and the inside of the neurone becomes more negative. Therefore, the membrane is less likely to reach threshold and an action potential is unlikely.

At an inhibitory synapse...

Summation

Temporal Summation Temporal summation occurs when two or more action potentials (nerve impulses) arrive in rapid succession along a single pre-synaptic neurone.

Spatial Summation Spatial summation occurs when two or more separate inputs arrive almost simultaneously from different pre-synaptic neurones. The individual pre-synaptic potentials add together.

Summation is the method of signal transduction between neurons, which determines whether or not an action potential will be triggered by the summation (adding together) of postsynaptic potentials.

Neuromuscular Junction and Muscle Fibres

Muscles always exist in antagonistic pairs where one

contracts whilst the other relaxes. E.g. The biceps and triceps

Skeletal Muscle – What Does It Look Like?

Microscopic structure of skeletal muscle.

Gross structure of skeletal muscle.

Thin filaments are made up of actin and tropomyosin.

Thick filaments are myosin.

Both together form myofibrils which bundle up to form muscle tissue.

H zone

A band

I band

No I or H zone when the muscle is contracted.

The Sliding Muscle Theory of Muscle Contraction

Contraction of Muscles

When an impulse reaches the neuromuscular junction and the neurotransmitter binds to the post-synaptic receptors, Ca2+ ions are released around the actin molecules.

These ions bind to calcium ion binding sites which causes tropomyosin to move and expose the myosin binding sites on the actin molecule.

ATP molecules are hydrolysed to ADP and inorganic phosphate. The energy released is transferred to the myosin heads which – with the ADP attached - move and bind to the exposed binding sites on the actin molecule forming a cross bridge. The ADP is released

ATP

ADP m

Pi

Contracted

Relaxed

Cross bridge cycleThe complete cycle of a myosin cross-bridge bending, binding, sliding and returning to its original position.

ATP and Photocreatine

ATP and Phosphocreatine provide energy for muscle fibres.ATPHydrolysis of ATP provides energy for movement of the myosin heads to form

cross bridges.ATP also provides energy for the reabsorption of Ca2+ ions by active transport

(after they have caused the movement of tropomyosin.)

PhosphocreatineIs stored in muscle and acts as a reserve supply of phosphate (needed to make

ATP). Phosphocreatine enables phosphate to immediately combine with ADP to regenerate the ATP used in muscle contraction.

This phosphocreatine store is replenished using phosphate from ATP when muscle is relaxed.

Slow and Fast Twitch Muscle Fibres

SLOW TWITCH MUSCLE FIBRES

FAST TWITCH MUSCLE FIBRES

Structure Lots of mitochondria, stores of myoglobin (oxygen store)

Thicker and more myosin, lots of enzymes for anaerobic respiration, phosphocreatine

Location Muscles such as calf muscles Muscles such as the bicep

General Properties

EnduranceLess powerAerobic respirationHigh fatigue resistance

Powerful, short-term contractionsIntense exerciseAnaerobic respirationLow fatigue resistance

Drugs at the Synapse

- If the drug has a similar shape to the neurotransmitter it will probably bind to the post-synaptic receptor and block impulse transmission.

- Some drugs will bind to acetylcholinesterase and stop it from breaking down the neurotransmitter, therefore the impulse will carry on being initiated (excitatory).

Don’t need to know specific drug action, however...

Why Have Nerve Impulses?

Simple reflexes avoid damage to the body, for example, when your hand flinches away from a hot object.

Nerve impulses can control heart rate which allows the heart to speed up when oxygen demand increases.

All receptors respond to specific stimuli e.g. Rod and cone cells result in a nerve impulse to the brain which produces images which allow us to see.

Control of Heart Rate

Controlled by a region of the brain called the medulla oblongata.The medulla oblongata has two centres:•Centre which increases heart rate = linked to sinoatrial node by sympathetic nervous system•Centre which decreases heart rate = linked to sinoatrial node by the parasympathetic nervous systemCHEMICAL and PRESSURE changes in the blood stimulate parts of the brain via receptors.

Chemoreceptors • Found in the wall of the carotid arteries. • They are sensitive to pH changes that results from CO2

concentration changes (exercise is a cause of increased CO2)

• When the chemoreceptors detect this change they increase the frequency of nerve impulses to the medulla oblongata

• This centre increases impulse frequency via the sympathetic nervous system to the sinoatrial node of the heart increased heart rate.

• The increases blood flow increases amount of CO2 removed at the lungs and therefore pH returns to normal.

• The chemoreceptors detect the change and reduce the impulse frequency back to normal levels.

Control of Heart Rate cont.

Pressure Receptors• Found in the wall of the carotid arteries and the aorta.• When blood pressure is higher than normal they send

nervous impulse to the medulla oblongata which decreases heart rate by sending impulses via the parasympathetic nervous system to the sinoatrial node of the heart; lowering the pressure back to normal.

• When blood pressure is lower than normal they send nervous impulse to the medulla oblongata which increases heart rate by sending impulses via the sympathetic nervous system to the sinoatrial node of the heart; raising the pressure back to normal.

Control of Heart Rate cont.

Pacinian CorpuscleAnother example of a receptor

is the Pacinian Corpuscle. Which is found all over the body and sends a nerve impulse to the brain when stimulated allowing us to have the sense of touch.

When the Pacinian corpuscle is exposed to pressure, stretch-mediated sodium channels become deformed (and open) leading to the establishment of a generator potential.

Pressure

The Eye

1= Bipolar Cell2= Cone3= Rod

RODS CONESMonochromatic Vision Colour Vision

Good Sensitivity Poor Sensitivity

Many rods connected to one bipolar cell poor acuity = poor resolution

Each cone is connected to one bipolar cell good acuity = good resolution

Is used for peripheral vision as found all over the retina.

Found mainly on the fovea, which means that can only detect images in centre of retina.

PRINCIPALS OF

HOMEOSTASISHomeostasis in mammals involves physiological control systems that maintain the

internal environment within restricted limits. (e.g. Body temperature at around 37°.)

Negative Feedback:Negative feedback systems maintain systems at a preset level by detecting deviations from that level

and initiating corrective mechanisms to restore it. e.g. Body temperature and blood glucose concentration are controlled by negative feedback.The body has separate mechanisms for controlling these deviations in each directions providing greater

control. e.g. Different areas of the brain for temperature loss and temperature gain

Positive Feedback:Positive feedback systems exaggerate a deviation from the preset and is often associated with the break

down of control systems.e.g. During hyperthermia, the negative feedback system breaks down and a positive feedback loop is

established meaning that the body’s core temperature continues to decrease.e.g. During childbirth, contractions of the uterus wall releases oxytocin which then increases the

contractions further until the child is born.

MAMMALIAN HORMONES... ...are substances that stimulate their target cells via the blood

system. This results in a slow, long-lasting and widespread response.

CHEMICAL MEDIATORS...

...are released from cells and only affect cells in the immediate vicinity. They are usually released by injured or infected cells. Their secretion causes blood vessels around the area to dilate (inflammation)

Histamine-Produced by mast cells in response to injury or allergen.-Causes capillaries to dilate and their walls to become more permeable which allows some of the plasma to leave the blood.-This increased permeability causes swelling and makes it easier for phagocytes to exit the blood and ingest the dead tissue and bacteria found in the wound.Prostaglandins-Produced by mast cells in response to injury or allergen.-Cause warmth, pain and redness around the injured area.-As well as causing vasodilation of arterioles, prostaglandins promote blood clotting which minimises blood loss from the wound and stop entry of microorganisms.

Homeostasis – Body Temperature Control

Increase in skin/blood temperatureNerve impulse sent to the heat loss centre of the hypothalamus via the autonomic nervous system.Vasodilation of skin arterioles which increases radiation of heat.Increased sweating which increases heat loss by evaporation.

Decrease in skin/blood temperatureNerve impulse sent to the heat gain centre of the hypothalamus via the autonomic nervous system.Vasoconstriction of skin arterioles which decreases radiation of heat.Stop sweating to avoid the heat loss through evaporation.Piloerector muscles contract, hairs stand up on body to insulate heat

The maintenance of body temperature and blood pH is important as too high or low a temperature/pH could denature the enzymes of the body or prevent them from working efficiently.

Ectotherms V Endotherms

• Homeotherms/Endotherms - These are organisms that that regulate their own body temperature internally using biological mechanisms. Their internal body temperature is independent of the external temperature. (Don't use the term 'warm-blooded').

• Poikilotherms/Ectotherms - These are organisms that cannot regulate their own body temperature internally. Their internal temperature fluctuates with the external temperature. (Don't use the term 'cold-blooded'). They use behavioural mechanisms to control their core body temperature.

Homeostasis – Blood Glucose Concentration

It is important to maintain a constant blood glucose concentration because:

-Too high plasma glucose conc. results in kidney malfunction and tissue dehydration.

-Too low plasma glucose conc. results in fatigue, paleness and possibly losing consciousness (coma).

- A deviation from healthy blood glucose conc. could result in excess water loss or gain by red blood cells due to the change in water potential.

-Cells around the body could shrink or burst if tissue fluid were to contain too much or too little glucose.

Blood glucose concentration is affected by many factors such as the body’s ability to produce insulin, the amount of exercise taken, alcohol

consumed, diet etc.

Glucose Glycogen

Insulin(Glycogenesis)

(Glycogenolysis)Glucagon

Maintained by two hormones; insulin and glucagon which are found in the Islets of Langerhans in the pancreas. Both hormones bind to liver cell receptor proteins which releases enzymes which catalyse the interconversion of glucose and glycogen.

Increase in glucose conc Detected by islet cellsα-cells decrease glucagon secretion

ᵝ-cells increase insulin

secretion.

...break down the glucose into glycogen. (Glycogenesis)

Decrease in glucose conc Detected by islet cellsα-cells increase glucagon secretion

ᵝ-cells decrease insulin secretion.

...which convert glycogen to glucose and this is released into the blood. (Glycogenolysis)

...both insulin and glucagon bind to receptor proteins on the surface of liver cells and through a cascade system activate enzymes which control the interconvertion of glucose and glycogen: Once they bind to the receptor, a G-protein is activated which turns ATP into cyclic AMP (the second messenger). This cyclic AMP then activates enzymes which...

The effect of other hormones on plasma glucose concentration

Adrenaline- Increases the breakdown of glycogen to glucose.- Increases the release of glucose into the

bloodstream to allow increased energy release in the muscles.

Thyroxin- Increases metabolic rate leading to an increase in

energy requirements.

Not on spec.

Diabetes

What is it?- Type 1 diabetes is where the ß cells in the islets of Langerhans are damaged

and cannot produce sufficient insulin so blood glucose conc. becomes erratic.

- Type 2 diabetes is where not enough insulin is produced by the ß cells AND the receptors on the liver cells become insensitive to it.

- Symptoms for both Type 1 + 2 diabetes include urinating more, fatigue, loss of weight and excessive thirst.

How can it be controlled?- Type 1 diabetes requires insulin being administered (by injection)- Type 2 can be treated with oral medication- Both types can be controlled by a carefully managed diet and regular

exercise.

Menstrual CycleFSH• The hormone FSH is secreted by the pituitary gland. FSH makes two things happen:• it causes an egg follicle to mature in an ovary• it stimulates the ovaries to release the hormone oestrogenOestrogen• The hormone oestrogen is secreted by the ovaries. Oestrogen makes three things happen:• It causes the repair of the lining of the uterus wall in preparation for the implantation of a

blastocyte.• it stops FSH being produced - so that only one egg matures in a cycle• it stimulates the pituitary gland to release the hormone LHLH (luteinising hormone)• The hormone LH is secreted by the pituitary gland.• The hormone LH causes the mature egg to be released from the ovary.Progesterone• Progesterone is a hormone secreted by the corpus luteum (left over after the egg is released

from the follicle) in the ovaries. • Progesterone maintains the lining of the uterus during the middle part of the menstrual cycle

and during pregnancy.

Teacher 2 – Genetic Control in Cells 3.5.6 3.5.8

Universal, non-overlapping, degenerate, base triplet code.

The genetic code

DNA triplet codes for same amino acid in all living organisms.

Each triplet is distinct from other triplets and only helps code for a single amino acid.

Each amino acid has several triplets which will code for it.

Three nucleotides which make up the code for a specific amino acid.

DNA STRUCTURE

Hydrogen bonds between the complementary bases hold the helix together.

Covalent bonds hold the sugar phosphate backbone together.

Anti-parallel strands

mRNA and tRNA STRUCTURE

• Both have Uracil (U) base instead of Thymine (T).

• Both single stranded instead of double stranded.

• Both have ribose sugar backbone rather than deoxyribose sugar backbone.

Transcription – In the nucleus

DNA helix is unwound by DNA helicase.RNA polymerase produces a pre-mRNA strand using

complementary base pairing and RNA nucleotides found in the nucleus. (The pre-mRNA is complementary to the anti-sense strand of DNA so therefore produces the protein that the sense strand codes for.)

(DNA helix re-winds.)Introns and promoter regions are removed from the pre-

mRNA which is then spliced back together to form mRNA.(and capped with a nucleotide which ribosomes can recognise.)

mRNA leaves the nucleus through the nuclear pores and enters the cytoplasm...

Translation – In the cytoplasm

mRNA reaches a ribosome.tRNA molecules that have complementary anti-

codons to the codons on the mRNA strand bind to those codons bringing their specific amino acid.

Peptide bonds form between the amino acids carried by the tRNA’s.

The ribosome moves along the mRNA strand one codon at a time and tRNA’s continuously deliver amino acids to the ribosome until a stop codon is reached and the polypeptide chain is complete.

(The amino acid chain then leaves the ribosome ready for modification.)

Overview of Transcription/Translation

DNA - AGT.CTT.GAC.ACT

mRNA – UCA.GAA.CUG.UGA(mRNA is spliced after removal of introns)

tRNA – AGU, CUU, GAC, ACU

Amino Acid sequence – Ser, Leu, Asp, Thr

Protein

Gene Mutation

Occur spontaneously during DNA replication and involve the deletion or substitution of bases.

Chance increased by mutagens such as carcinogenic chemicals and radiation.

Some mutations results in a changes amino acid sequence (different or inactive protein).

Others do not change the amino acid sequence because of the degenerate nature of the code.

Stem Cells

Totipotent cells all genes in this unspecialised cell have the potential to be expressed. A zygote has totipotency. Able to grow / divide rapidly.

During development a totipotent cell translates only 3-5% of its DNA and becomes specialised.

PLANTS Many mature plant cells maintain totipotency and have the ability to develop in vivo into whole plants or into plant organs under correct conditions. This is why taking ‘cuttings’ can result in new plants.During micropropagation tiny samples of plant tissue can be grown on an agar plate with the correct nutrients and hormones to form explants.

Only a few totipotent cells remain in the adult human body - stem cells.

These are found in the bone marrow.These can be used to treat genetic disorders- the

stem cells, when under the correct conditions can form new tissue if humans have damaged their own. Bone marrow transplants are used for the treatment of leukaemia etc.

Stem Cells - continued

Genes which control cell divisionProto-oncogenes control the rate of cell

division.

..... but mutates to become....

Oncogenes stimulate the cells to divide too rapidly causing a tumour to develop.

Tumour suppressor genes slows cell division

..... but mutates to become....

Inactive, so cell division continues and a tumour develops.

Regulation of transcription and translation

Genes are only expressed once a specific transcriptional factor moves from the cytoplasm into the nucleus. (These are usually proteins.)

The transcriptional factor binds to the promoter region near the required gene.

RNA polymerase binds to the DNA transc. factor complex and is activated causing it to move away from the complex and along the gene.

The RNA polymerase now transcribes the strand of DNA. (The gene is now being expressed.)

Oestrogen –Its effect on transcription

Oestrogen diffuses through the plasma membrane of a cell and binds with an oestrogen receptor forming a complex (alters shape of the receptor).

The complex (transcriptional factor) moves from the cytoplasm to the nucleus where it binds to the promoter region and activates the transcription of a target gene.

There are drugs with a similar shape to oestrogen which block the receptors and stop transcription. This is a treatment for breast cancer sufferers.

oestrogen

oestrogen

oestrogen

oestrogen

cytoplasm nucleus

siRNA

A short double-strand of RNA which interferes with (represses) the expression of a specific gene.

Post-transcriptional interference.

mRNA extracted from cells where desired DNA is found.

Reverse transcriptase enzyme

Free DNA nucleotides

From mRNA Using Restriction Endonucleases

Creating DNA / DNA fragments

The DNA is cut at specific palindromic recognition sequences using restriction enzymes.

PCR

Used to make multiple copies of DNA fragments. In a PCR machine which constantly heats up and cools down to break and reform hydrogen bonds.

Template DNA, Primers,

DNA polymerase enzyme, DNA nucleotides

37ºC

72ºC

95ºC

In VIVO and In VITRO cloning of genes

In VITROThe use of PCR in cloning DNA

fragments. Can be used for paternity tests/crime investigations etc.

☺PCR is fully automated which makes it a quick and relatively cheap method of DNA cloning.☺Billions of copies of a small

DNA sample can be made.

In VIVOThe use of restriction enzymes and ligases

to insert DNA fragments into a vector (virus or liposome) which are then transferred into host cells.

These transformed host cells can be grown and therefore clone the desired DNA fragments.

‘Sticky ends’ are left by the restriction endonuclease which allow the exposed bases pairs to anneal to other strands with complementary ‘sticky ends’.

☺Delivers the gene, already in the organism- good for making products e.g. Insulin.

Genetic FingerprintingAn organisms genome contains many non-coding repetitive base

sequences which are very unlikely to be repeated in another individual.DNA fragments are cloned through PCR and can be analysed to find genetic

relationships such as a paternity test, to diagnose a genetic disease or to find genetic variation in a population.

Genetic fingerprinting is also used in forensic science to match hair/blood samples to suspects and in agriculture to produce genetically superior crops. Animal breeding can also be supplemented with genetic manipulation to produce animals with desirable characteristics.

Unethical to change the genotype of an animal: some changes can result in a decrease in their quality of life e.g. cows with huge udders for increased milk production.

The medical benefits of using recombinant plasmid technology and virus vectors outweigh the ethical issues, as the quality of life of the sufferer could be greatly improved.

Gene Therapy

Germ-Line Gene TherapyReplacing or supplementing defective gene in a

fertilised egg.

Somatic-Cell Gene TherapyTargeting affected tissue (so not passed on to next

generation) by introducing additional gene.

Effect is short-lived as cells treated will eventually die off.Can induce immune response.Not effective if a characteristic is controlled by more than one

gene.

The Use of Vectors somatic-cell gene therapy

Genes that code for useful substances, such as hormones, enzymes and antibiotics, are often transferred into micro-organisms, which then produce large quantities of these substances. The gene is cut out from the DNA of the donor organism using a restriction endonuclease enzyme. This cuts out the relevant section of the organism's DNA, leaving sticky ends, that will enable the gene to be inserted into a small circular piece of bacterial DNA called a plasmid. Plasmids are often used as vectors to take the selected gene into bacterial cells. The same restriction endonuclease is used to cut the plasmid. This leaves complementary sticky ends to which the selected gene can be attached. The sticky ends of the gene and the open plasmid are joined together by ligase – the new transformed plasmid is called a RECOMBINANT PLASMID. These plasmids are then introduced into the target organism.

Virus VectorsA virus is made harmless and the desired gene is

introduced to it. The virus is then breathed inby the sufferer of a genetic disease and the virus implants the gene into their infected cells. This DNA is incorporated into their cellsand the gene is activated.

Liposome VectorsLiposomes are tiny spheres or phospholipids and other chemicals.

Because of their small size they can pass easily through plasma membranes and carry the desired gene with them.

How do we know which bacteria now contain the desired gene?

GENETIC MARKERS in the plasmids, such as genes that confer antibiotic resistance, enable genetic engineers to identify bacteria that have successfully taken up the selected gene.

The insertion of the new gene can split the gene for antibiotic resistance so any bacteria which were once resistant but are now killed by the antibiotic have recombinant plasmids.

Transformed cells are selected and cloned.

How do we know which bacteria now contain the desired gene? Cont.

USING DNA PROBESIncubate the recombinant plasmids with bacteria, then dilute

the bacterial suspension and culture on an agar plate, The different bacteria will colonise different areas of the Petri dish.

Blot the Petri dish with filter paper and break open the cells on the filter paper and split the DNA strands.

Incubate with specific DNA probes which have a complementary base sequence to part of the desired DNA – these will bind to the desired base sequence.

Using an X-ray (which highlight where the DNA probes are on the sample) the position on the filter paper corresponds with where the colonies of interest are on the original Petri dish.

Determining the Base Sequence of the Desired Gene.Once located, the base sequence of the gene can be determined by...DNA SequencingThe ‘Sanger Method’ (the chain terminator technique) is used to find out the exact order of the nucleotide in the DNA section.

DNA nucleotides and a small quantity of ‘T terminator’ nucleotides

The DNA fragments which want sequencing

Primer to start DNA synthesis

DNA polymerase

DNA nucleotides and a small quantity of ‘A terminator’ nucleotides

The DNA fragments which want sequencing

Primer to start DNA synthesis

DNA polymerase

DNA nucleotides and a small quantity of ‘G terminator’ nucleotides

The DNA fragments which want sequencing

Primer to start DNA synthesis

DNA polymerase

DNA nucleotides and a small quantity of ‘C terminator’ nucleotides

The DNA fragments which want sequencing

Primer to start DNA synthesis

DNA polymerase

A

T

G

C

The ‘Sanger Method results in many DNA fragments. In the ‘A tube’ the DNA polymerase will form DNA strands with the DNA nucleotides but at some point will pick up an ‘A terminator’ nucleotide which will block further nucleotides from forming the strand.

In the ‘A tube’ at the end there will be...

In the ‘T tube’ at the end there will be...

In the ‘C tube’ at the end there will be...

In the ‘G tube’ at the end there will be...

The sequence can then be worked out from the position of the fragments on the agar plate.

Restriction MappingUsing a restriction enzyme on large DNA sections provides fragments. The

more fragments, the more cuts were made by the restriction enzymes. The number of cuts equal the number of recognition sites on that DNA strand.

If these fragments are put through gel electrophoresis then the relative distance between cut sites is determined.

A restriction map is made of the original DNA.

More Recent Developments...The specification says:

“ ” Candidates should understand the principle of these methods [DNA sequencing and restriction mapping]. They should be aware that methods are continuously updated and automated.

One of these developments involves tagging the nucleotides with fluorescent dyes. These are all run on the same electrophoresis gel lane. As the nucleotides migrate they pass a laser that detects the dye. The whole system is automated and can be linked to a PCR machine for small samples.

Genetic Counselling

Most human diseases results from a mutated genes or from genes which are useful in one context but not in another.e.g. Sickle cell anaemia causes a decrease in oxygen concentration capacity but people with the disease cannot contract malaria.

Genetic counselling enables people to make decision about themselves and offspring:

The person is genetically screened - using specific DNA probes - for the presence of

genetic conditions. Through genetic counselling the person can decide whether to

have pre-emptive treatment if for example they have oncogenes which will lead to

tumours. Partners who are both carriers for a genetic disease can decide if the risk of passing

on the gene is too great to reproduce.

Enzymes in Genetics

• RNA polymerase – joins RNA nucleotides (transcription).

• DNA polymerase – joins DNA nucleotides (copying DNA).

• Ligase – joins 2 strands of DNA (S-P backbone).• Reverse transcriptase – joins DNA nuc’s to make

copy of mRNA.• Restriction endonucleases – cut DNA in specific

places – create sticky ends.