BiologyUnit 5

AQA

Nervous & Hormonal Communication Receptors detect stimuli and effectors produce a

response:◦ Receptors can be cells or proteins on cell surface membranes

◦ Effectors include muscle cells & cells found in glands

Nervous system sends information as electrical impulses:◦ Sensory Neurone – Transmits impulses from receptor to CNS

◦ Motor Neurone – Transmits impulses from CNS to effector

◦ Relay Neurone – Transmits impulses between Sensory & Motor Neurones

Stimulus Receptor CNS Effector Response Peripheral Nervous System:

◦ Made up of neurones that connect CNS to rest of body

Somatic Nervous System:◦ Controls conscious activities e.g. running

Autonomic Nervous System:◦ Controls unconscious activities e.g. digestion

◦ Sympathetic: - stimulates effectors/speeds things up

◦ Parasympathetic: - inhibits effectors/slows things down

Nervous & Hormonal Communication

Hormonal system is made up of glands & hormones:◦ Group of specialised cells that secrete a useful substance

◦ Hormones are ‘chemical messengers’ mostly proteins or peptides

Glands stimulated by:◦ Change in concentration of a specific substance

◦ Electrical impulse

Hormones diffuse directly into the blood Hormones usually trigger a response in the target

cells

They’re specific to one particular stimulus They can be cells, or proteins on cell surface

membranes

How they work:

◦ When nervous system receptor at resting state there’s a difference in voltage between inside & outside of cell

◦ Resting potential is when a cell is at rest

◦ When a stimulus is detected, cell membrane becomes excited & more permeable. This allows ions to move in & out of cell.

◦ This alters the potential difference (PD) of cell

◦ Change in PD is called the generator potential (GP)

◦ Bigger stimulus = more ion movement = bigger GP

◦ If the GP is big enough & reaches threshold, a AP is produced

Receptors

Receptors in Skin & Eye Pacinian Corpuscles – Pressure Receptor in Skin:

◦ They’re mechanoreceptors – detect mechanical stimuli e.g. pressure & vibrations

◦ Contain the end of a sensory neurone – a sensory nerve ending

◦ Ending wrapped in layers of connective tissue – lamellae

◦ When stimulated the lamellae are deformed & press on the sensory nerve ending

◦ This deforms the stretch-mediated Na+ channels in the sensory neurone’s cell membrane

◦ Allows Na+ to move into the cell creating a GP

◦ If the GP reaches the threshold then an AP is produced

Photoreceptors – Light Receptors in Eye:◦ Light enters through pupil – amount of light let through controlled by

iris muscles

◦ Light rays are focused onto the retina by the lens

◦ Retina contains the photoreceptor cells

◦ Fovea - lots of photoreceptor cells

◦ Nerve impulses travel down the optic nerve to brain, where it leaves the eye is the blind spot

Receptors in Eye Photoreceptors convert light to an electrical impulse:

◦ Light enters eye, hits photoreceptors & absorbed by light-sensitive pigments

◦ Light bleaches pigments, causes chemical change – membrane more permeable to sodium

◦ If GP reaches threshold an AP is produced & is sent down the bipolar neurone

◦ Bipolar neurones connect photoreceptors to the optic nerve

◦ Two type of photoreceptor: Rods – Found in peripheral part of retina – Give information in black & white Cones – Found packed together in fovea – Three types of cone – red, green & blue sensitive

◦ Sensitivity: Rods very sensitive to light. Many rods join one neurone, so many weak GP’s combine

to reach threshold and trigger an AP Cones less sensitive to light. One cone joins one neurone, so more light is needed to

reach the threshold

◦ Visual Acuity: Rods have low visual acuity, many rods join one neurone. This means light from two

objects close together can’t be told apart Cones have high visual acuity because each cone joins one neurone and the cones are

tightly packed together. When light from two different points hit, two APs are produced. Allows us to see two points

Neurones Resting potential – difference in voltage across the membrane when at

rest Resting potential maintained & created by Na+/K+ Pumps & K+ Channels

Stimulus:◦ Bigger stimulus fires more frequently

◦ Causes Na+ channels to open

◦ Membrane more permeable to Na+

◦ Na+ diffuses into neurone

◦ Inside of neurone more negative

Depolarisation:◦ If PD reaches -55mV (threshold), more Na+ open

◦ More Na+ diffuses into neurone

Repolarisation:◦ At +30mV Na+ channels close and K+ open

◦ K+ diffuse out of neurone

Hyperpolarisation:◦ K+ slow to close

◦ Too many K+ diffuse out of neurone

◦ More –ve than resting potential

Resting Potential:◦ Na+- K+ pump returns membrane to rest

Refractory Period:◦ Makes sure no overlaps or travel in one direction

K+ OpenNa+ Close

ThresholdNa+ Open

K+ Close

Refractory Period

Factors Affecting Speed of Impulse Myelination:

◦ Some neurones have a myelin sheath – electrical insulator

◦ Made up of Schwann cells

◦ Between Schwann cells are nodes of Ranvier

◦ Neurone’s cytoplasm conducts enough electrical charge to depolarise next node

◦ Impulse ‘jumps’ from node to node – saltatory conduction

Axon Diameter:◦ Faster with larger diameters – less resistance

◦ Depolarisation reaches other parts of neurone quicker

Temperature:◦ Ions diffuse faster at higher temperature

◦ Up to 40°C – after that the proteins denature

Motor Neurone

Sensory Neurone

Myelinated Axon

Dendrites

Cell Body

Motor End Plate

Synapses Presynaptic neurone has a swelling – ‘synaptic knob’ Synaptic knob contains vesicles – filled with

neurotransmitter When AP reaches end of neurone causes neurotransmitter

to be released into synaptic cleft Neurotransmitter diffuses across cleft & binds to receptor Triggers AP, Muscle Contraction or Hormone Secretion Neurotransmitter removed from receptor by enzymes Cholinergic Synapse:

◦ AP arrives at knob

◦ Stimulates voltage-gated Na+ channels to open

◦ Ca2+ diffuse into knob

◦ Causes vesicles to fuse with presynaptic membrane

◦ Vesicles release ACh (Acetyl Choline) in cleft – exocytosis

◦ ACh binds to specific receptors on postsynaptic membrane

◦ Na+ channels open on postsynaptic membrane

◦ Na+ influx produces an AP

◦ ACh removed from receptor by AChE and reabsorbed into presynaptic membrane

More Synapses Neuromuscular Junction:

◦ Synapse between motor neurone & muscle cell

◦ Similar to cholinergic synapse, but a few differences

◦ Post-synaptic membrane has lots of folds forming clefts, containing AChE

◦ Post-synaptic membrane has more receptors

◦ AP from motor neurone always triggers a response in a muscle cell

Neurotransmitter are Excitatory or Inhibitory:◦ Excitatory depolarise the post-synaptic membrane, making it fire an AP if

threshold is reached e.g. ACh

◦ Inhibitory hyperpolarise the post-synaptic membrane preventing an AP being fired e.g. GABA

Summation:◦ Effect of neurotransmitter released from many neurones is added together

◦ Spatial: Many neurones connect to one neurones Small amount of neurotransmitter from each can be enough to reach

threshold & produce AP If some release inhibitory neurotransmitter then total effect might not

produce an AP

◦ Temporal Two or more impulses arrive in quick succession from same pre-synaptic

neurone

Drugs Affecting Action of Neurotransmitter

Same shape as neurotransmitter, mimic their action so more receptors are activated

Block receptors so they can’t be activated by neurotransmitter, less receptors activated

Inhibit enzyme that breaks down neurotransmitter, receptor is blocked by neurotransmitter

Stimulate release of neurotransmitter from pre-synaptic membrane

Inhibit release of neurotransmitter from pre-synaptic membrane

Muscles Skeletal muscles (striated, striped or voluntary muscles) Made up of muscle fibres Cell membrane of muscle fibre cells is the Sarcolemma Bits of sarcolemma fold inwards across the muscle fibre and stick into

the sarcoplasm (muscle cell’s cytoplasm) These folds are called transverse (T) tubules & help spread impulses

throughout sarcoplasm Network of internal membranes called Sarcoplasmic Reticulum Sarcoplasmic reticulum stores Ca2+ needed for muscle contraction Muscle fibres are multinucleate (contain many nuclei) Muscle fibres have lots of long, cylindrical organelles called Myofibrils Myofibrils:

◦ Contain thick & thin filaments

◦ Thick = made of myosin

◦ Thin = made of actin

Muscle Contraction

Myosin has globular heads With a binding site for ATP & actin Tropomyosin & troponin found between actin filaments, helps filaments

slide over each other At rest actin-myosin binding site blocked by tropomyosin, held in place

by troponin AP depolarises sarcolemma, spreading down T tubules to sarcoplasmic

reticulum Sarcoplasmic reticulum releases Ca2+ into sarcoplasm Ca2+ binds to troponin causing it’s change shape Pulls tropomyosin out of actin-myosin binding site, exposing site Bond between myosin head & actin creating a actin-myosin cross bridge Ca2+ also activates ATPase, providing energy Energy released moves myosin head, pulling actin molecule ATP provides energy to break cross bridge Myosin head reattaches to different binding site, creating new cross

bridge When excitation stops Ca2+ leave binding site on troponin & moves into

sarcoplasmic reticulum by AT

More Muscle Contraction Aerobic Respiration:

◦ Most ATP produced via oxidative phosphorylation in mitochondria

◦ Long periods of low-intensity exercise

Anaerobic Respiration:◦ ATP made rapidly by glycolysis

◦ Produces pyruvate which converts into lactate

◦ Lactate builds up in muscles causing fatigue

◦ Short periods of high-intensity exercise

ATP-Phosphocreatine (PCr) System:◦ ATP produced by phosphorylating ADP, adding a phosphate group from PCr

◦ PCr stored inside cells

◦ Quick production of ATP

◦ PCr runs out after a few seconds

◦ ATP-PCr is anaerobic & alactic (no lactate formed)

Slow-Twitch Fibres:◦ Aerobic – Red Colour – high levels of Myoglobin

Fast-Twitch Fibres:◦ Anaerobic – White Colour – low levels of Myoglobin

Control of Heart Rate SAN generates impulses controlling HR Unconsciously controlled by Medulla Stimuli are detected by baroreceptors & chemoreceptors:

◦ Baroreceptors in Carotid Arteries – stimulated by Blood Pressure

◦ Chemoreceptors in Carotid Arteries – monitor O2, CO2 & pH in blood

◦ Chemoreceptors detect change in pH

◦ More CO2 = More Carbonic Acid = Lower pH

Stimulus Receptor

Neurone & Neurotransmitt

er

Effector Response

High BPBaroreceptor

Impulse to medulla, along parasympathetic to SAN

Cardiac Muscle

HR slows BP lowers

Low BP Impulse to medulla, along sympathetic to SAN

HR increases BP increases

Low CO2

Chemoreceptor

Impulse to medulla, along parasympathetic to SAN

HR slows CO2 level returns to

norm

High CO2Impulse to medulla, along

sympathetic to SAN

HR increases CO2 level returns to

norm

Reflexes Involuntary rapid response to a stimuli

Thermoreceptors in skin detect heat stimulus Sensory neurone carries impulse to Relay Relay to Motor Motor sends impulse to effector (e.g. biceps) Effector muscle contracts stopping hand being damaged

If relay neurone involved you can override the reflex e.g. leave your hand on the heat

Responses in Animals Tactic response (taxes):

◦ Organisms move towards or away from a directional stimulus

◦ Phototaxis – Light

◦ Chemotaxis – Chemicals

◦ Aerotaxis – Air (O2)

◦ Geotaxis - Gravity

Kinetic response (kineses):◦ Organisms’ movement is affected by the intensity of the stimulus

Cells communicate with other cells with Chemical Mediators:◦ Chemical mediator is a chemical messenger that acts locally

◦ Similar to hormones

◦ Chemical mediators can be secreted from cells not just from glands

◦ Target cells near to where mediator is produced – local response

◦ Only travel a short distance – quicker response

Histamines:◦ Stored in mast cells & basophils. Released in response to infection or

injury. Allows more immunity cells to move in & out of blood to the area

Prostaglandins:◦ Produced by most cells of the body. Involved in inflammation, fever, BP

regulation & blood clotting

Responses in Plants Tropism – Plants Growth Response to an External Stimulus +ve tropism = growth towards the stimulus Phototropism:

◦ Growth in response to Light

◦ Shoot grow towards light +vely phototropic

◦ Roots grow away from light -vely phototropic

Geotropism:◦ Growth in response to Light

◦ Shoots are -vely geotropic, grow upwards

◦ Roots are +vely geotropic, grow downwards

Gibberellin is a growth stimulus flowering & seed germination Auxins stimulates growth of shoots by cell elongation Auxins inhibit growth in roots IAA is an important Auxin – diffuses over short distances &

via phloem over long distances

Root

Root Shoot

Shoot

Homeostasis Homeostasis involves control systems that keep your internal

environment roughly constant Temperature:

◦ If too high enzymes denature, enzymes molecules vibrate, breaks H-bonds, shape of active site is altered

◦ Optimum - 37°C

pH:◦ pH too high or low, enzymes denature

◦ Optimum usually – 7 but specialist enzymes can work higher or lower

Glucose:◦ Too high H2O potential of blood is more –ve. H2O moves out of cells by

Osmosis

◦ Too low, cells unable to function properly due to lack of energy

Negative Feedback:◦ Mechanism that returns the level to normal but only works between certain

limits. If change is too big then effectors may not be able to counteract it

◦ Multiple negative feedback mechanisms give more control

Positive Feedback:◦ Mechanism that amplifies the change from the norm, effectors respond to

further increase level away from norm.

◦ Blood Clotting – Platelets become activated – trigger more platelets to be activated

Controlling Body Temperature

Mechanism of Heat Loss:◦ Vasodilation –arteriole diameter near skin increases – warm blood passes

near skin

◦ Increased Sweating – H2O lost by evaporation, body heat used to evaporate H2O

◦ Lowering of Body Hair – less insulating layer – hair erector muscles in skin relax

◦ Behavioural - sheltering in the shade/burrows

Mechanisms of Heat Gain:◦ Vasoconstriction – arteriole diameter near skin decreases – blood passes

under insulating fat

◦ Shivering – muscles involuntarily contract – produces metabolic heat

◦ Hairs Stand Up – hair erector muscles contract – traps layer of air next to skin

◦ Hormones – releases adrenaline & thyroxine – increase metabolic rate – more heat produced

◦ Less Sweating

◦ Behavioural – basking in sun / huddling / sheltering from wind

Ectotherms e.g. reptiles Endotherms e.g. mammals

Control temp by changing their behaviour

e.g. basking in sun

Control temp internally by homeostasis & behaviour

Internal temp depends on external temp

Internal temp less affected by external temp

Activity depends on external temp

Activity independent on external temp

Variable metabolic rate High metabolic rate – produces heat

Hypothalamus Controls body temperature in mammals Heat Loss Centre:

◦ Responds to rise in body temp

Heat Gain Centre:◦ Responds to fall in body temp

Receives information about both internal & external temperature

Information produced by Thermoreceptors

Internal temperature:◦ Thermoreceptors in hypothalamus detect blood temperature

External temperature:◦ Thermoreceptors in skin detect skin temperature

Thermoreceptors send impulses to hypothalamus along autonomic nervous system

To effectors from hypothalamus along autonomic nervous system

Hormonal Control of BGC Insulin:

◦ Lowers BGC when too high

◦ Produced by Beta cells in the Islets of Langerhans (pancreas)

◦ Binds to receptors on cell membrane of liver & muscle cells

◦ These cells are more permeable to glucose, cell takes up more glucose

◦ Insulin activates enzyme converts glucose to glycogen

◦ Cells store glycogen in cytoplasm – energy source

◦ Glycogenesis = glucose glycogen

◦ Insulin increases rate of respiration of glucose, especially in muscle cells

Glucagon:◦ Raises BGC when too low

◦ Produced by Alpha cells in Islets of Langerhans (pancreas)

◦ Binds to receptors on membrane of liver cells

◦ Activates enzyme breaks down glycogen into glucose

◦ Glycogenolysis = breaking down glycogen

◦ Gluconeogenesis = forming glucose from non-carbohydrates

◦ Glucagon reduces rate of respiration of glucose in cells

BGC – Blood Glucose Concentration

Control of BGC Adrenaline:

◦ Hormone secreted by adrenal glands

◦ Secreted if BGC is low

◦ Binds to receptors on liver cells Activates glycogenolysis – glycogen glucose Inhibits glycogenesis – glucose glycogen

◦ Activates glucagon secretion

◦ Inhibits insulin secretion

◦ Adrenaline & Glucagon bind to receptors activating Adenylate Cyclase (enzyme)

◦ This enzyme converts ATP into a chemical signal a ‘second messenger’

◦ Second messenger is Cyclic AMP (cAMP)

◦ cAMP activates a chain of reactions breaking down glycogen into glucose (glycogenolysis)

Diabetes:◦ Type 1:

No insulin produced, BGC stays high – hyperglycaemia, treated with insulin injection

◦ Type 2: Don’t produce enough insulin or don’t respond to insulin Treated by controlling simple carbohydrate intake

Menstrual Cycle

Lasts 28 days Follicle developing in the ovary Ovulation – egg being released Uterus lining thickens, to support implanted egg Corpus luteum develops from follicle remains

FSH (Follicle-Stimulating Hormone) – stimulates follicle to develop

LH (Luteinising Hormone) – stimulates ovulation & corpus luteum development

FSH & LH from Pituitary Gland

Oestrogen – Stimulates uterus lining thickening Progesterone – maintains uterus lining thickening Oestrogen & Progesterone from Ovaries

1. High [FSH] in Blood:

FSH stimulates follicle development

Follicle releases oestrogen

FSH stimulates oestrogen to be released by ovaries

2. Rising [Oestrogen]:

Oestrogen stimulates uterus lining thickening

Oestrogen inhibits FSH

3. [Oestrogen] Peaks:

High [oestrogen] stimulates FSH & LH production

4. LH Surge:

Ovulation stimulated by LH

LH stimulates follicle corpus luteum

Corpus luteum releases progesterone

5. [Progesterone] Rises:

Progesterone inhibits FSH & LH

Maintains uterus thickening

If no embryo implants, corpus luteum breaks down. No more progesterone produced

6. Falling [Progesterone]:

FSH & LH increase (not inhibited by progesterone)

Uterus lining not maintained so it breaks down - menstruation

-VE & +VE Feedback - Hormones Negative Feedback: Example One:

◦ FSH stimulates Ovary to release Oestrogen

◦ Oestrogen inhibits release of FSH

After FSH has stimulated Follicle development, -ve feedback keeps [FSH] low, so no more follicles develop

Example Two:◦ LH stimulates Corpus Luteum, which produces Progesterone

◦ Progesterone inhibits release of LH

-ve feedback makes sure no more Follicles develop when Corpus Luteum is developing

Also makes sure Uterus Lining no maintained if no Embryo implants

Positive Feedback: Example:

◦ Oestrogen stimulates Pituitary Gland to release LH

◦ LH stimulates Ovary to release Oestrogen

◦ Oestrogen further stimulates to release LH

High Oestrogen concentration triggers +ve feedback to make Ovulation happen

DNA It’s a large polymer of repeating Nucleotides. G & A Large Bases (Purines)

C & T Small Bases (Pyrimidines)

A - T, C - G are the complementary pairs Small % of DNA are genes, the rest is junk

(VNTRS) used to create a DNA fingerprint DNA molecules found inside nucleus, but

organelles for protein synthesis are found in cytoplasm

DNA too large to move out of nucleus Sections are copied into RNA which then moves

to ribosomes

A – AdenineG – GuanineC – CytosineT (U) – Thymine(Uracil in RNA)

P – Phosphate GroupS – SugarN – Nitrogen Base (Varies)

Gene Loci

Exon

Intron

A & T have 2 H bondsG & C have 3 H bonds

RNA – Single Stranded Messenger RNA (mRNA):

◦ Made during transcription in the nucleus

◦ Carries genetic information from nucleus to the cytoplasm

Transfer RNA (tRNA):◦ Clover-shaped

◦ Every molecule has a specific anticodon at one end

◦ Amino Acid binding site at other end

◦ Found in cytoplasm where it’s involved in translation

DNA mRNA tRNA

Shape Double Stranded Helix

Single-Stranded Single-Stranded

Sugar Deoxyribose Ribose Ribose

Bases A T C G A U C G A U C G

Other… 3 Bases = 1 Codon 3 Bases = 1

Codon3 Bases = Anticodon

or AA Binding Site

Protein Synthesis Transcription: RNA polymerase attaches to DNA double-helix H-bonds between helix break One strand is template for mRNA copy RNA polymerase attaches free RNA nucleotides to template strand RNA nucleotides then form mRNA molecule RNA polymerase moves along strand of DNA H-bonds reform in DNA, back to a double-helix Stops making mRNA if reaches a stop signal (specific base sequence) mRNA moves out through nuclear pore & attaches to ribosome

Splicing: Pre-mRNA contains Introns & Exons Introns are removed during splicing In the nucleus Only DNA that codes for AA’s remains

Translation: mRNA attaches to ribosome & tRNA carries AA to ribosomes tRNA anticodon molecule attaches to mRNA codon (specific base

sequence) 2nd tRNA attaches to next mRNA codon 2 AAs joined by peptide bond 1st tRNA molecule moves away leaving it’s AA 3rd tRNA attaches to next codon, it’s AA bonds to previous 2 AAs Process continues producing a polypeptide chain until a stop signal

on mRNA Polypeptide chain moves away from ribosome

Protein Synthesis

Genetic Code

It’s non-overlapping, degenerate & universal

The genetic code is the sequence of codons in mRNA which code for AAs

Each codon is read in sequence, each codon is read separately

Degenerate:◦ Some AAs are coded for by more than one base triplet

Some codons are used to start & stop protein production

Same codons code for AAs in all organisms

Regulation of Transcription & Translation Transcription Factors:

◦ They move from the cytoplasm into the nucleus

◦ Bind to specific DNA sites near start of target gene

◦ They control the rate of transcription

◦ Activators – increase the rate

◦ Repressors – decrease the rate

Oestrogen◦ Binds to an oestrogen receptor and acts as a transcription factor

◦ Forms an oestrogen-oestrogen receptor complex which moves into the nucleus

◦ It binds near the start of the target gene

◦ Complex can act as an activator or a repressor

◦ Depends what type of cell & the target gene

siRNA (small interfering RNA):◦ Short, double-stranded RNA molecules

◦ Their bases are complimentary to sections of target gene

◦ Interferes with transcription & translation

◦ Affects translation through RNA interference: In cytoplasm, siRNA & proteins bind to target mRNA Proteins cut mRNA into sections so it can’t be translated Prevents expression of specific genes as translation cannot occur

Mutations Change in the base sequence of DNA Caused by:

◦ Errors during DNA replication

◦ Mutagenic agents

Substitution:◦ One base is substituted for another – causes frame shift

Deletion:◦ One base is deleted

But not all mutations affect order of AAs:◦ Degenerate nature of DNA

◦ Substitution may not affect AA order but deletion always will

Mutagenic Agents:◦ UV radiation, Ionising radiation, some chemicals & viruses

◦ Act as a base – chemicals called base analogues can substitute for a base during DNA replication

◦ Altering bases – some chemicals can delete or alter bases

◦ Changing DNA structure – some radiation can change shape of DNA

Genetic Disorders & Cancer

Hereditary Mutations:◦ Some mutations can cause genetic disorders

◦ Some mutations can increase risk of developing certain cancers

◦ If a gamete containing a gene mutation is fertilised it becomes a hereditary mutation

Acquired Mutations:◦ Mutations that occur after fertilisation

◦ If mutations occur in genes controlling cell division, can cause uncontrollable cell division

◦ Produces a tumour – mass of abnormal cells

◦ Tumours invade & destroy surrounding tissues are Cancers

◦ Two genes control cell division: Tumour-Suppressor Genes – mutation causes protein not to be produced uncontrollable cell

division Proto-Oncogenes –mutation causes it to be overactive cells to divide uncontrollablyProto-OncogenesTumour-Suppressor Genes

Stem Cells Able to mature into any body cell Stem cells are unspecialised cells Stem cells found in embryo and some adult tissues Stem cells that can mature into any body cell are totipotent

cells Totipotent cells only found in early life of embryo After this point they lose ability to become any cell

Totipotent found in plants:◦ Mature plants have them where they grow e.g. roots & shoots

◦ All plants stem cells are totipotent

◦ Can be used for tissue cultures – cell placed in sterile growth medium – produces new plant

Totipotent become specialised because they only translate & transcribe part of their DNA◦ Certain genes are expressed others are turned ‘off’

Making DNA Fragments Using Reverse Transcriptase:

◦ Most cells contain 2 copies of each gene but contain many mRNA copies

◦ Enzyme converts RNA DNA

◦ DNA produced from RNA is cDNA (complementary DNA)

Using Restriction Endonucleases:◦ Enzymes recognise palindromic sequences of nucleotides

◦ DNA sample incubated with the specific restriction endonuclease

◦ Can leave sticky ends allowing the DNA fragment to anneal to another DNA molecule if they have a complementary base match

Using PCR (Polymerase Chain Reaction):◦ Mixture with Primers, DNA sample, Free Nucleotides & DNA Polymerase

◦ Primer – short piece of DNA complementary to bases at start of fragment required

◦ DNA mixture heated to 95°C to break H-bonds in double strand

◦ Cooled to 60°C so primers can anneal to strand

◦ Mixture heated to 72°C so DNA polymerase can work

◦ DNA polymerase forms a new strand complementary to the template strand

◦ Two new copies are formed, one cycle of PCR complete

◦ Cycle starts again

◦ Each PCR cycle doubles the amount of DNA

Gene Cloning In Vitro:

◦ Where the gene copies are made outside of a living organism using PCR

In Vivo:◦ Where the gene copies are made within a living organism

In Vivo:◦ Gene Inserted into a Vector:

DNA fragment inserted into vector DNA Vector – something used to transfer DNA into a cell Vector DNA cut open using restriction endonuclease DNA ligase joins sticky ends of DNA fragment to vector DNA, Ligation New combination of bases in DNA – recombinant DNA

◦ Vector Transfers the Gene into Host Cells: If a plasmid vector used, host cells won’t easily take in the Plasmid vector

& it’s DNA With a bacteriophage vector, it will infect the host bacterium by injecting

it’s DNA into it Host cells that take up the vector containing the gene are Transformed

◦ Identifying Transformed Host Cells: Marker genes inserted into vector along with gene Host cells grown on agar – creates colony of cloned cells Marker gene can code for antibiotic resistance – agar contains antibiotic –

only transformed cells with survive Can also code for fluorescence – will show up under UV light

Organisms with altered DNA are called transformed organisms

Genetic engineering - recombinant DNA technology

Genetic Fingerprinting Genomes contain repetitive, non-coding DNA sequences Number of repeats of a sequence can be compared between

individuals

Gel Electrophoresis:◦ PCR is used to make copies of sample

◦ Primers bind to each end of the repeat

◦ Fluorescent tag added to DNA fragments – visible under UV light

◦ DNA mixture placed into a well in the gel

◦ Gel covered in a buffer that conducts electricity

◦ Electric current passed through the gel, DNA moves towards +ve electrode

◦ Small fragments move further

Used to determine:◦ Relationships

◦ Variation

◦ Medical Diagnosis

◦ Forensic Science

Locating & Sequencing Genes

Look for genes using DNA Probes & Hybridisation:◦ DNA probes locate gene or see if DNA contains a mutated gene

◦ DNA probes are short strands of DNA, they have a complementary base sequence to part of the target gene

◦ DNA probe will bind to target gene & can be detected if it is labelled (fluorescent or radioactive)

◦ DNA sample digested with restriction enzymes & separated by gel electrophoresis

◦ DNA fragments transferred onto a nylon membrane & incubated with labelled DNA probe

◦ Membrane exposed to UV or X-ray & bands will be visible

Restriction Mapping:◦ Restriction enzymes cut labelled DNA into fragments

◦ Gel electrophoresis produces bands

◦ Compare DNA with different restriction enzymes

Gene Sequencing:◦ Mixture of DNA template, polymerase, primer, nucleotides & Fluorescently

labelled modified nucleotides

◦ Each tube contains A*, G*, C* or T* & undergoes PCR

◦ Different length fragments produced depending on where the modified fragments bind to the DNA e.g. ACTACG*, ACTACGATG* in tube with G*

DNA Probes in Medical Diagnosis

They can screen for mutated genes e.g. Sickle-cell Anaemia:

◦ Probe labelled to look for a specific gene

◦ Or Probe used as part of microarray, which can screen many genes at the same time DNA Microarray is a glass slide with microscopic spots of different DNA probes

attached in rows Sample of labelled human DNA washed over the Array Any matches with the probes will stick to the array Array washed to remove excess DNA Array under UV light shows any labelled DNA Any spot that fluoresces contains that specific gene

Results of screening used to decide what treatment to use

Gene Therapy Involves altering the defective genes inside cell to treat

genetic disorders & cancers Depends if it is caused by mutated dominant or double-

recessive alleles Allele inserted into cells using a vector Somatic Therapy:

◦ Altering the alleles in body cells, particularly ones most affected by disorder

◦ Doesn’t affect sex cells so offspring could still inherit disease

Germ Line Therapy:◦ Alters sex cells

◦ Offspring will not be affected by the disease

◦ Illegal at the moment