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ESA 1 Revision > MGD > Flashcards

MGD Flashcards

1
Q

What are the functions of the Golgi Body?

A

Export of proteins

Membrane synthesis

Detoxification reactions

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2
Q

What is the function of the cytoplasm?

A

Metabolism of carbohydrate, amino acids and lipids

Fatty acid synthesis

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3
Q

What is the function of lysosomes?

A

Cellular digestion with hydrolytic enzymes.

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4
Q

What are the functions of mitochondria?

A

ATP synthesis

Beta oxidation of fatty acids

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5
Q

What is the function of the endoplasmic reticulum?

A

Lipid and steroid synthesis (smooth), protein export. membrane synthesis, detoxification

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6
Q

What is the function of the nucleus?

A

DNA synthesis and repair. DNA is in the form of chromatin

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7
Q

What is the function of the nucleolus?

A

RNA synthesis and ribosome assembly

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8
Q

What is the function of the plasma membrane?

A

Transport of ions and small molecules, cell morphology and movement

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9
Q

What is the function of a ribosome?

Where are they found?

A

Protein synthesis (free in cytoplasm or attached to RER)

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10
Q

What is a bacterial cell wall made of?

A

Peptidoglycan

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11
Q

How many pairs of chromosomes are there in a human cell?

A

23

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12
Q

How is DNA present in a bacterial cell?

A

In a circular loop

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13
Q

What type of ribosomes are found in:

Eukaryotes?

Prokaryotes?

A

Eukaryotes: 80S

Prokaryotes: 70S

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14
Q

What does solubility depend on?

A

The ability to make hydrogen bonds

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15
Q

How are monomers joined together to form macromolecules?

A

Covalent bonds

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16
Q

How are macromolecular complexes held together?

A

Non-covalent interactions

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17
Q

What is a hydrogen bond?

A

An electromagnetic attraction between a hydrogen atom bonded to an electronegative atom, and another electronegative atom

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18
Q

Define the term hydrophilic:

A

Polar molecules which can interact with water forming hydrogen bonds, and therefore dissolve in water

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19
Q

Define the term hydrophobic

A

Non polar molecules which can’t interact with water molecules, and are therefore insoluble in water

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20
Q

What is a hydrophobic interaction?

A

Non polar regions/molecules cluster together in order to repel water.

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21
Q

What is an amphipathic molecule?

A

A molecule with both polar and non polar ends

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22
Q

What is pH?

A

A measure of the concentration of H+ ions in solution

-log[H+]

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23
Q

What are van der Waals forces?

A

forces between two atoms in close proximity.

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24
Q

How to strong and weak acids differ?

A

Strong acids dissociate completely in solution, whereas weak acids only partially dissociate.

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25
Q

What is Ka (and pK), and what do different values of it mean?

A

Ka is the acid dissociation constant, a measure of how strong or weak an acid is, -log to give pK. Strong acids have a large Ka (low pK), and weak acids have a small Ka (large pK).

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26
Q

What is a buffer?

A

A mixture of a weak acid and its conjugate base, which resists changes in pH on addition of a small amount of acid or base.

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27
Q

Henderson Hasselbach equation

A

pH = pKa + log [A-]/[HA]

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28
Q

When pH>pK…

A

The deprotonated form dominates

(Higher pH, less H+ in solution, so acid dissociates to give more)

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29
Q

When pH<pk...>
</pk...>

A

The protonated form dominates

(There is lots of H+ in solution so the acid is less likely to dissociate)

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30
Q

When pH = pK…

A

The amount of acid and its conjugate base is equal

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31
Q

How can amino acids be classified?

A

Aliphatic/aromatic

Non polar, polar uncharged, postive, negative

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32
Q

What are the 2 negatively charged amino acids?

A

Aspartate and glutamate

(acidic, lose a proton)

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33
Q

What are the 3 positively charged amino acids?

A

Lysine, aspargine, histidine

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34
Q

What is a peptide bond?

A

A covalent bond between the O of a Carboxyl group and the H of an amino group

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35
Q

What are 4 key features of peptide bonds?

A

All atoms in the bond are on the same plane.

Formed in a condensation reaction.

No rotation.

Trans orientation.

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36
Q

What is a zwitterion?

A

A neutral molecule with both a positive and negative charge

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37
Q

What is the optimum blood pH? and what range must it stay within?

A

7.4

Range: 7.38 to 7.42

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38
Q

Define pI

A

Isoelectric point: The pH value at which the protein has no overall charge.

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39
Q

Properties of acidic proteins

A

Lots of negatively charge amino acids

Low pI

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40
Q

Properties of basic proteins

A

Lots of postive amino acids

High pI

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41
Q

If pH is higher than isoelectric point…

A

NEGATIVE ion

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42
Q

If pH is lower than isoelectric point…

A

POSTIVE ion

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43
Q

Define primary structure of a protein

Bonds involved?

A

The linear amino acid sequence

Covalent

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44
Q

Define secondary structure of a protein

Bonds involved?

A

The local spatial arrangement of the polypeptide backbone.

Hydrogen bonds

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45
Q

Define the tertiary structure of a protein

Bonds involved?

A

The 3D arragement of all atoms in a polypeptide, the folding of secondary structures.

Hydrogen, ionic, disulphide, hydrophobic interactions, van der waals

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46
Q

Define quarternary structure of a protein

Bonds involved?

A

The 3D arrangement of multi sub unit proteins, with more than one polypeptide chain.

Same as tertiary

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47
Q

What are the major features of an alpha helix?

A

RIght handed helix

  1. 6aa per turn
  2. 54nm pitch

Carboxyl of one aa bonded to amine 4 aas away

Ala and Leu, small hyrdophobic aas, strong helix formers

Gly and Pro are helix breakers

R groups point outwards

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48
Q

What are the key features of a beta pleated sheet?

A

Extended conformation

0.35nm between adjacent aas

Parallel/antiparalell

Alternate side chains in opposite directions

B strands make B sheets

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49
Q

What factors cause protein denaturation? Explain

A

Heat: increased vibrational energy.

pH: alters ionisation states of aas.

Detergents/ organic solvents: disrupts hydrophobic interactions, breaking tertiary structure

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50
Q

What is an amyloidoses?

A

Altered conformation of a protein promotes conversion of an existing protein into a misfolded one, forms insoluble aggregates.

Usually highly ordered with large proportion of beta sheets.

Inter chain assembley stabilised by hydrophobic interactions.

Stain: CONGO RED

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51
Q

What is a fibrous protein?

A

Only primary and secondary structure

Insoluble

Support function

No tertiary

Single type of repeating secondary

Long strands/sheets

Collagen

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52
Q

What is a globular protein?

A

Up to quaternary structure

Soluble

Catalysis and regulation

Compact

Several types of secondary

Lots of tertiary

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53
Q

Explain the physiological role of myoglobin

A

Single subunit protein

One Haem group

So can bind one oxygen molecule

Stored in the muscles, used when oxygen is in high demand, acts as a store

Hperbolic oxygen binding

No cooperativity

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54
Q

Explain the physiological role of haemoglobin

A

Tatrameric protein

2 alpha and 2 beta subunits

Four Haem groups

4 molecules of oxygen

Sigmoidal/cooperative binding

Picks oxygen up in the lungs and delivers it to the tissues for respiration

Some carbon dioxide transport

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55
Q

Why does oxygen need to be transported by proteins?

A

Non polar

Can not diffuse far in water

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56
Q

Describe the affinity of myoglobin for oxygen

A

Very high affinity for oxygen

Will only release oxygen with pO2 is very low

Hyperbolic curve

No cooperativity

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57
Q

Describe the oxygen binding properties of haemoglobin

A

Cooperative binding

Sigmoidal binding curve

Low affinity T state = tense

High affinity R state = relaxed

Transition between the two gives the sigmoidal curve

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58
Q

Describe the properties of the haem groups

A

Porphyrin ring and an Fe atom bound to 4 N atoms of the ring

Bound to protein via histidine residue

Fe sits slightly below the ring, O2 binding causes it to move into plane

Changes the conformation of the bound polypeptide

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59
Q

What is cooperativity in Haemoglobin?

A

Low affinity T state and high affinity R state

Oxygen binding changes the conformation in all 4 sub units, promoting the binding of subsequent O2 molecules

The sigmoidal curve makes it a more efficient carrier, more sensitive to smal differences in pO2.

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60
Q

What is the effect of 2,3-bisphosphoglycerate on the binding of oxygen to Hb?

A

Regulates oxygen binding

Binds to Hb

Decreases oxygen affinity

Curve shifts to the right

1 BPG per tatramer of Hb

[BPG] increases at high altitudes promoting O2 release to tissues

A BPG isomer is produced in glycolysis, so O2 is released more readily in areas that are metabolically active

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61
Q

What is the effect of carbon dioxide and H+ ions on the affinity of Hb for O2?

A

Both bind to haemoglobin

More acidic

Lowers the affinity of Hb for oxygen

Sites of low pH and high carbon dioxide

BOHR EFFECT

Curves shifts to the right

Release of oxygen

Metabolically active tissues produce lots of H+ and CO2

Ensures delivery of oxygen is coupled to demand

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62
Q

What is the effect of CO on the binding of oxygen to Hb?

A

Binds tightly to Hb, 250x more readily than oxygen

Prevents O2 binding

Blocks O2 transport

Fatal when COHb is more than 50%

Increases the affinity of unaffected sub units for oxygen

High affinity state: releases less oxygen to tissues

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63
Q

What inheritance pattern does sickle cell anaemia have?

What mutation causes it?

A

Autosomal recessive

Single nucleotide subsitution

Missense

A changes to a T

Glutamate to Valine

Beta subunit

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64
Q

What effect does the mutation in sickle cell anaemia have?

A

Blood disorder

Valine creates a hydrophobic pocket in the beta sub unit

Causes deoxygenated Hbb to polymerise

Distortion of RBCs into sickle cell shape

Stress to cell membrane, premature cell lysis

Lifespan of only 30 days compared to 120 days

Blocks microvasculature

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65
Q

What are thalassaemias?

A

Group of haemoglobinopathies

Genetic disorders

Imbalance between alpha and beta subunits

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66
Q

What is alpha thalassaemia?

A

Descreased or absent alpha chains

Different severity levels as multiple copies of alpha gene are present

Beta chains can form stable tetramers with increased affinity for oxygen

Onset before birth

(as alpha subunits in fetal Hb a2y2)

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67
Q

What is beta thalassaemia?

A

Decreased or absent beta chains

Excess alpha chains

Can’t form stable tetramers

Symptoms appear after birth

As B globin only in adult Hb a2B2

3 - 6 months after birth, lose fetal Hb

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68
Q

Explain the effects of enzymes on chemical reactions?

A

Lower activation energy needed for a reaction to occur

Bidning of substrate to an active site

Increases local concentration of reactions

Stabilised formation of a high energy transition state

Increase rate of reaction

Non convalent binding of substrate, highly specific

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69
Q

Describe some properties of enzymes

A

Do not effect the equilibrium of the reaction

Proteins

Some require cofactors: inorganic ions

Some need coenzymes: organic carriers of reaction groups

Highly specific

Unchanged after reaction

Active site: a few amino acids supported by a scaffold, cleft or crevices, to exclude water

Substrate changes enzyme shape slightly, induced fit

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70
Q

How does reaction rate vary as a function of enzyme and substrate concentration?

A

An enzyme E, combines with a substrate S, to make an enzyme substrate complex, ES, to break down to form free enzyme and product, P.

From this can derive michaelis menten equation, which describes how reaction velocity varies with substrate concentration

Increase substrate concentration, reaches maximal velocity, rectangular hyperbola

Increase enzyme concentration, directly proportional to reaction rate

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71
Q

What is the international unit of enzyme activity?

Define Km

Define Vmax

A

Unit: the amount of enzyme that produces one micromole of product per minute

Km: the substrate concentration that with give half the maximal rate; Vmax. Also equates to affinity of enzyme for substrate. Low Km = high affinity and vice versa

Vmax: the maximum velocity/rate of an enzyme catalysed reaction, when the enzyme is saturated with substrate

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72
Q

Interpret kinetic data for enzyme catalyed reactions:

Vo:

Lineweaver Burk Plot:

How to interpret Km

How to interpret Vmax

Slope

A

Vo is the initial reaction velocity

Lineweaver Burk Plot:

X axis: 1/[S], intercept is Km

Y axis: 1/V, intercept is Vmax

Slope is Km/Vmax

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73
Q

Effects of inhibitors on enzyme kinetics:

No inhibitor:

Irreversible inhibitor:

Reversible inhibitors -

Competitive:

Non-competitive:

A

No inhibitor: reaches Vmax

irreversible: covalently bonded

Competitive: binds at active site, affects Km but not Vmax. Adding enough substrate will overcome effect.

Non competitive: binds away from active site, affects Vmax, but not Km, cannot be overcome

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74
Q

List the major regulatory mechanisms that control enzyme activity:

A

Allosteric: enzymes with more than one subunit, the binding of substrate to one site can inhibit or activate and enzyme. Cooperativity. Eg phosphofructokinase, activated by AMP, Fructose 2 6 bisphosphate. Inhibited by ATP, citrate, H+

Substrate and product concentration: substrate availability, co enzyme availability, accumulation of product can inhibit forward reaction, eg glucose 6 phosphate inhibits hexokinase

Covalent modification: attachment of groups covalently via amino acids. Attachement of phosphates via kinases and removal by phosphatases.

Proteolytic activation: secreted as inactive zymogen and cleaved by proteases to active enzyme, eg trypsinogen to trypsin

Changes in the amount of enzyme: regulation of enzyme synthesis and protein degradation

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75
Q

What is an enzyme cascade?

A

When enzymes activate other enzymes

The number of affected molecules increases in the enzyme cascade

Number of affected molecules increases geometrically

Allows quick amplification of an initial signal by several orders of magnitude quickly

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76
Q

Give examples of zymogens and their active enzymes

Also give their activators and where they are found

A

Pepsinogen to pepsin, activated by pH in the stomach

In the pancreas:

Trypsinogen to trypsin activated by enteropeptidase

Chymotrypsinogen to chymotrypsin activated by trypsin

Procarboxypeptidase to carboxypeptidase activated by trypsin

Proelastase to elastase activated by trypsin

a1 antitrypsin deficiency linked with emphysema

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77
Q

Describe allosteric enzymes

A

Multi sub unit enzymes that contain more than one active site

SIgmoidal relationship, cooperative

2 different comformations: tense and relaxed

Substrate binding to pme subunit makes progressive binding easier

PFK, step 3 of glycolysis:

Activators: AMP, fructose-2,6-bisphosphate

Inhibitors: ATP, citrate and H+

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78
Q

What do allosteric activators and inhibitors do?

Effect on curve

A

Activators: increase activity of enzyme, curve shifts to the left, increases proportion of enzyme in R state

Inhibitors: decrease activity of enzymes, curve shifts right, increases proportion of enzyme in the T state

T converts to R upon binding, equilibrium shifts to the right

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79
Q

What do kinases do?

A

Transfer the phosphate group from ATP

To the -OH group of Ser, Thr, Tyr

Big negative charge added

Changes the conformation, substrate binding

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80
Q

What do phosphatases do?

A

Removed phosphate groups through hydrolytic activity

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81
Q

Define the term zymogen

How are they activated?

What sort of enzymes are commonly made as zymogens?

Examples?

A

Inactive precursors of enzymes

Activated by removal of part of the polypeptide chain

Many proteases are synthesised in this way: makes for safe transport

Blood clotting factors

Apoptosis: procaspase to caspase

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82
Q

How can enzymes be regulated by changes in the amount of enzyme?

A

Change in transcription rate, ribosome rate

Regulated protein degradation, tag with the small protein ubiquitin

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83
Q

Explain how the activation of the clotting cascade leads to the formation of fibrin

A

Intrinsic pathway, damaged endothelial lining of blood vessels promotes binding of factor 11a

Extrinsic pathway: trauma releases factor 3, tissue factor

Postivie feedback from thrombin, further activation of cascade to allow clot formation from very small amount of initial factor.

Factor 10 bring both together, the 2 THROMBIN, then 1 FIBREIN, stabilised by calcium

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84
Q

What is the role of calcium and vitamin K in the clotting cascade?

A

Release of calcium

y carboxyglutamate, Gla domains, carboxylated on clotting factors IX and X

Need vit K as co factor

Target appropriate sites for activation

Activates factors

Cleaved proteolytically

Brings together clotting factors

Calcium needed for factors 1, 2, 10, 9 and 7

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85
Q

How is a clot formed?

A

Fibrinogen to fibrin

Fibrinogen: 2 sets of tripeptides a, B, y, joined at N terminal by disulphide bonds. N terminal regions highly negatively charged, prevent fibrinogen aggregation

Prothrombin binds Ca2+ on gla residues to thrombin

Thrombin cleaves fibrinogen A and B feet

C terminal ends of B and y (globular) interact with cleaved N terminal ends of A and B to form fibrin clot

Cross bridges between lysine and glutamine residues, catalysed by transglutaminase

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86
Q

What do these blood clotting factors do?

III?

VIII?

XI?

A

III: tissue factor, extrinsic pathway, trauma activates

VIII: haemophilia A

XI: intrinsic pathway

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87
Q

Describe regulation of the blood clotting cascade?

A

Inactive zymogens present at low concentrations, so to prevent accidental clot activation

Dilution of clotting factors by blood flow and removal by the liver

Amplification of signal

Localisation of clotting factors to site of damage: factor with Gla binds to endothelial cells which are damaged

Feedback activation by throbmin, enhances conversion of V, VIII and XI to active forms

Termination by many processes: Digestion of factors by proteases, Va and VIII a by protein C, activated by thrombin

Plasmin degrades clots. Converted from plasminogen by t-PA and streptokinase

Specific inhibitors antithrombin 3, enhanced by heparin binding, does not act on thrombomodulin bound thrombin (on endothelial cells)

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88
Q

What are the components of RNA and DNA molecules?

A

Nitrogenous bases

Pentose sugar

Phosphate, ester bond between it and C5 of sugar

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89
Q

What bases are present in DNA?

What bases are present in RNA

A

C, G, A, T

C, G, A, U

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90
Q

What are the types of nitrogenous base?

What is the difference between them?

Which bases are which type?

How are base pairs formed?

A

Purine and Pyrimidine

Purines have 2 rings, Pyrimidines have 1

A and G are purine. C, T and U are pyrimidine

Base pairs are formed between purines and pyrimidines, by hydrogen bonds

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91
Q

What is the charge of a DNA molecule? Why?

A

Negative

From phosphate group

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92
Q

What sugar is in RNA?

What sugar is in DNA?

A

Ribose

2-deoxyribose

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93
Q

How many strands in

RNA?
DNA?

A

RNA: 1

DNA: 2

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94
Q

What is a nucleoside?

What is a nucleotide?

A

N side, sugar and a base

N tide, sugar, base and phosphate

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95
Q

How are DNA and RNA sequences typically read?

A

5’ to 3’

5’ starts with phosphate, 3’ is hydroxyl

Top strand is always 5’ to 3’

Left to right

Bases given letters

Duplex structure include complementary antiparallel strand

Hydrogen bonds denoted by dotted lines

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96
Q

Explain the polarity of a DNA or RNA chain

A

Polynucleotides are nucleotides linked covalently by phosphodiester bonds

Each single strand has polarity

5’ end is free phosphate

3’ end is free OH

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97
Q

Which nitrogenous bases pair together?

How many bonds between each?

A

C and G, 3 H bonds

A and T/ U, 2 H bonds

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98
Q

How do the hydrogen bonds form between bases?

A

O atoms in carbon ring are elctronegative

N group is dipole positive

Interaction between electronegativity of oxygen and dipole of nitrogen

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99
Q

How are duplex structures formed in RNA and DNA?

How are RNA stem loops formed?

A

Complementary antiparallel strands

DNA-DNA/RNA-RNA/RNA-DNA

Stable/temporary/invivo/in lab

DNA double helix

RNA, strand loops back on itsself, one side is antiparallel, H bonds between complementary bases

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100
Q

Describe the key features of the DNA double helix

A

2 independent polymers

Complimentary and antiparallel

Top strand 5’ to 3’, vice versa

Space between base pairs 0.34 nm

Purines and pyrimidines planar and unsaturated

Major grooves of exposed bases

and minor grooves

in structure of sugar phosphate backbone

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101
Q

How is eukaryotic DNA condensed in chromosomes?

A

Chromatin is present in interphase

NUCLEOSOMES: beads on a string, DNA wound twice around histone core, which is postiively charged

This is EUCHROMATIN, expressed genes, can replicate in interphase

SOLENOIDS: Nucleosomes are coiled to form 30nm fibres, highly condensed, can not be replicated, mitotic chromosomes

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102
Q

What phase of interphase is DNA replicated in?

A

S

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103
Q

Describe the process of DNA replication

A

INITIATION:

Strands unzipped by HELICASE. Origin of replication recognised, each strand initiated by PRIMASE. Specific proteins must interact with DNA and recruit DNA polymerase (can only extend from 3’ ends)

ELONGATION:

Each strand replicated by DNA polymerase, 5’ to 3’. Leading strand, continuously. Lagging strand, discontinnously in Okazaki fragments. Fragments joined by DNA ligase from OH group to phosphate group covalently.

TERMINATION:

Replication forks join, leading moves towards lagging.

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104
Q

What sort of replication is DNA replication?

A

Semi-conservative

one of the original DNA strands is in each of the new molecules, in each of the daughter cells

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105
Q

How does DNA polymerase work?

A

Uses deoxyribonucleoside triphosphate molecules as substrate

Base sequence determined by complementary template strand

Driven by pyrophosphate hydrolysis

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106
Q

How do helicase enzymes work?

A

Use ATP energy to unwind the DNA helix

Allow DNA replication

Around the replication fork helicases reform the DNA helix

Multiple replication forks along dsDNA

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107
Q

Describe the process and role of the cell cycle

A

G1 phase: growth, cell content replication, preparation for replication

S: DNA replication

G2: double check chromosomes and repair

MITOSIS and CYTOKINESIS, used to maintain and repair the body

Several key checkpoints to prevent improper cell division

Outside: G0, cell cycle arrest, for example nerve cells. Some may go back to G1

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108
Q

Describe the process of mitosis

A

All genetic info replicated in the S phase of interphase

Prophase: breakdown of nuclear membrane, chromosomes condense, kinetochore of spindle fibres attaches to centromere

Metaphase: chromosomes in X shape line up on metaphase plate

Anaphase: centromeres divide, spindle fibres contract, sister chromatids to opposite poles

Telophase: nuclear membrane reforms, chromosomes decondense, spindle disappears, cleavage of cytoplasm, cytokinesis

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109
Q

How to chromosome exist before mitosis, after replication?

A

Identical sister chromatids, joined by centromere

X shape

2 p arms and 2 q arms

Telomere at end of chromosome arm, prevents degradation

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110
Q

Describe the role of mitosis

A

Diploid cell

1 round of replication, 1 round of division

Cell division for somatic cells

Needed for tissue growth and early development

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111
Q

Describe the role of meiosis

A

Diploid cell, one round of replication, 2 rounds of division

4 haploid cells

Specialised cell division for germ line cells

2n to n

Production of eggs and sperm

Maintains constant chromosome number, diploid zygote

Generates genetic diversity from crossing over in prophase 1, and random assortment in metaphase 1

Produces 4 sperm and 1 egg, with polar bodies

112
Q

Describe the process of meiosis

A

Prophase 1: nuclear membrane disappear, chromosomes condense, bivalent forms, crossing over to form chiasmata

Metaphase 1: homologous pairs line up at metaphase plate randomly, bivalent. Independent assortment.

Anaphase 1: Homologous pairs pulled apart as spindle fibres contract. Nuclear membrane reforms.

Telophase 1: cytokinesis

Prophase 2: new spindle develops

Metaphase 2: chromosomes line up on metaphase plate

Anaphase 2: centromere divides, chromatids pulled to opposite ends by spindle

Telophase 2: chromatids lengthen and are indistinct, spindle disappears, nuclear membrane reforms, cytokinesis

113
Q

How common are mistakes in meiosis?

A

1/3 of all miscarriages

114
Q

What is genotype?

A

The genetic make up of an individual

Either as a whole

Or one specific genetic locus

Genes

115
Q

What is a phenotype

A

All observable characteristics of an individual

Expressed trait

As a result of the genetic make up

Of one or more specific gene loci

Expressed proteins

116
Q

Explain how environmental factors have an effect on both phenotype and genotype

A

For example

UV changes genotype to cause cancer

Unhealthy food changes phenotype, fat

Environmental factors:

Radiation

Mutagens

Chemicals that affect cell growth

Diet

Lifestyle

117
Q

What is a gene?

A

A unit of heredity

A length of DNA on a chromosome that codes for a protein

118
Q

What is an allele?

A

An alternative form of a gene

Each individual has two alleles of every gene

Can either be the same or different

119
Q

What are the different patterns of inheritance?

A

AUTOSOMAL:

Dominant

Recessive

SEX LINKED:

X linked dominant

X linked recessive

Y linked

120
Q

Explain autosomal dominant inheritance

A

Heterozygotes affected

Someone affected in every generation, all affected have an affected parent

Males and females equally affected

Rare disease in homozygotes: usually fatal

50% chance child is affected

Examples: huntingtons, neurofibromatosis 1, Marfan’s, familial hypercholesterolaemia IIa

121
Q

Explain autosomal recessive inheritance

A

Homozygotes only affected

Can skip generations, can seem to come out of nowhere

Males and females equally affected

Heterozygotes unaffected, carriers

2 heterozygotes, 25% chance affected offspring

2 homozygotes, only affected offspring

Examples

Cystic fibrosis, sickle cell disease, Tay Sachs

122
Q

Explain X linked recessive inheritance

A

Hemizygous males, always affected

Homozygous females affected rare

More common in males

Heterozygous female carrier, 50% chance of affected sons

Affected males can’t give trait to sons

Every affected male, heterozygous carrier mother

Every affected female, carrier mother, affected father

Daughters of affected males heterozygous

Examples: Haemophilia A, reg green colour blindness, Duchenne muscular dystrophy

123
Q

Explain Y linked inheritance

A

Rare

When gene is located on Y chromosome

Inherited directly from father to son

124
Q

Define dominance

A

Phenotypic trait is dominant when it occurs in heterozygotes and homozygotes

125
Q

Define recessive

A

When a phenotypic trait is only expressive in the homozygote

126
Q

Define Co dominance

A

When both allele in a pair are expressed and neither is dominant

127
Q

Describe complementation

A

more than one gene can be involved in producing a phenotype

EG ALBINISM

Therefore a child of two affected parents can be unaffected if parents have different defective genes

a1a1A2A2 x A1A1a2a2

CHILD

a1A1a2A2

128
Q

Describe the basis of co inheritance of certain traits

A

Genes on the same chromosome are linked, not independtly assorted at meiosis

Genes on different chromosomes are not linked, independently assorted in meiosis

If they are close together they are tightly linked, far apart, almost behave as unlined

Lots of genes are inherited on the same chromosome, a few from crossing over

Recombination frequency depends on the distance between genes

129
Q

What is a genetic map?

How does recombination frquency enable mapping of genes?

A

The arrangement and distance between genes on a chromosome deduced from studies of recombination

The frequency of recombanation between two loci gives information with respect to how close the two loci are

1 map unit = 1% recombination

Mapping is more accurate when genes are close together

130
Q

What is transcription?

Where does it occur?

What are the 3 phases?

What does it need to occur?

A

The process by which DNA is copied into an RNA message

The nucleus

Initiation, elongation, termination

Needs RNA polymerase, NTPs and a DNA template to occur

131
Q

Generally what happens in each phase and in processing afterwards?

A

Initiation: promotor recognition and binding

Elongation: transcription by RNA polymerase II

Termination: sequence dependent termination of RNA growth

Post translational processing from pre mRNA to mature mRNA

132
Q

What happens in initiation of transcription?

A

Initiation code is recognised: TATA box, 5’ to 3’

Transcription factors bind here, attracting RNA polymerase

Promoter recognition occurs, the promotor is an area upstream of the ORF which regulates gene expression, includes secquences for binding of transcription factors, RNA polymerase and regulatory factors

RNA polymerase seperates the DNA strands for the RNA nucleotides to bind along the template strand.

133
Q

What happens in elongation in transcription?

A

RNA polymerase travels along the template strand picking up base pairs and copying them onto a complementary RNA strand

Reads 3’ to 5’ on template, builds a 5’ to 3’ strand,

identical to coding DNA

134
Q

What happens in termination of transcription?

A

End of mRNA production

Dependent on the sequence

135
Q

What post transcriptional processing occurs to convert pre mRNA to mature mRNA?

A

CAPPING: 5’ methyl guanine cap. Bonded with 5’ - 5’ triphosphate linkage to stabilise the mRNA. Prevents degradation and has a role in translation.

TAILING: polyadenylation. Stop codon that cleaves mRNA, then 3’ end, lots of adenine nucleotides are added, protection against degradation. Up to 200 As.

SPLICING: premature product of mRNA has introns and exons. Introns which are non coding are removed. Cut by spliceosome proteins which recognise specific sequences. In PKU splicing is wrong

136
Q

Define

Exons

Introns

A

Exons are coding portions of DNA

Introns are non coding portions of DNA

137
Q

What are:

Endonucleases?

Exonucleases?

A

Endonucleases break within the polynucleotide, non specific or specific

Exonucleases degrade the polynucleotide from the 5’ or 3’ end

138
Q

What is translation?

Where does it occur?

What does it need to take place?

A

The process by which the genetic code carried by mRNA is read to produce a sequence of amino acids

Occurs in the cytoplasm

Needs: ribosomes, activated amino acyl tRNAs, mRNA substrates

139
Q

What are the three stages of translation? Briefly summarise each

A

Initiation: AUG codon recognition, binding of methionyl tRNA ribosome formation

Elongation: translating, N to C chain growth with addition of amino acyl tRNAs

Termination: stop codon recognition, dissociation of ribosome

140
Q

Describe initiation in translation

A

40S subunit with Met-tRNA binds to 5’ cap of tRNA

Anticodon on tRNA binds to AUG

60S subunit then binds

Release of GDP, initiation factors and cap binding proteins

141
Q

Describe elongation in translation

A

Met-tRNA occupies the P site

Another aminoacyl-tRNA enters the ribosome to occupy the A site

Needs GTP

Methionine forms a peptide bond with the adjacent amino acid

PEPTIDYL TRANSFERASE: release of water

The tRNA in the P site is now uncharged.

It leaves and the ribosome moves along, translocation

142
Q

Describe termination in translation

A

Stop codon is read on the mRNA

UAA, UAG or UGA

No tRNAs that can bind to these codons

Releasing factor pushes out mRNA

Peptide and tRNA are hydrolysed

Protein released into cytoplasm

Carboxy terminal is last

143
Q

What does a gene contain as well as the information for the protein?

A

Sequences necessary for expression

promotor

terminator

introns

144
Q

How is a tRNA activated?

A

uncharged tRNA

amino acid attaches, activating it

Charged

Created aminoacyl tRNA

Uses ATP

145
Q

Describe how the amino acids are coded for on the tRNA

A

tRNA anti codon matches mRNA codon

Inosine base is aspecific

WOBBLE POSITION

Degenerate code

146
Q

What are the 3 types of RNA?

Explain each

A

mRNA: messenger

RNA polymerase II, 2%, 100,000s of kinds due to splicing, few copies of each present

tRNA: transfer

RNA polymerase III, 15%, 100 kinds, very many copies of each

rRNA: ribosomal

RNA polymerase I, >80%, few kinds, many copies of each

147
Q

What is the function of a ribosome?

What kinds are found in eukaryotes and prokaryotes?

How do they read the genetic code?

A

Used to bind to the mRNA in translation in protein synthesis and provides the location for tRNA

Eukaryotic ribosomes: 80S: 60S and 40S subunits

Prokaryotic ribosomes: 70S: 50S and 30S subunits

Read in triplets with no overlapping and no gaps

148
Q

How do heterochromatin and euchromatin appear on an electron micrograph?

A

Heterochromatin: DARK

Euchromatin: LIGHT

149
Q

Describe the nature of the genetic code

A

4 letter DNA language

20 letter protein language of amino acids

Triplet code to create 20 amino acids

43 = 64 possibilites

Non overlapping and commaless, no gaps

5’ to 3’ read through

Degenerative, more than one code per amino acid

150
Q

What are the implications of the degeneracy of the genetic code?

A

Substitution of bases can lead to a different primary sequence

This can affect the tertiary structure of the protein

Mutation that affects a stop codon can cause it to be longer

Or mutation to create a stop codon: shorter

Some mutations are silent/neutral

Wobble tRNA with iosine

151
Q

How is gene expression in bacteria different to that in humans?

A

SImpler promotors

Different transcription factors

One one type of RNA polymerase

Coupled transcription/ translation

No post transcriptional processing

Short lived mRNAs

Simpler ribosomes

Distinctive translation initiation mechanism

Different translation factors

152
Q

How are differences in human and bacteria gene expression exploited clinically?

Specific examples of antibiotics inhibiting protein synthesis

A

Bacteria have simpler ribosomes: 30S subunit attacked

Streptomycin inhibits mRNA reading

Tetracycline blocks ribosome A site

Chloramphenicol prevents peptidyl transferase action

Erythromycin prevents translocation of peptidyl tRNA from A to P ste

153
Q

What are some types of mutation?

A

POINT: single base change

SILENT: change in base that specifies the same amino acid

MISSENSE: different amino acid coded

NONSENSE: produces a stop codon

INSERTION: addition of one or more bases

DELETION: loss of one or more bases

FRAMESHIFT: not a multiple of 3 bases deleted or added

154
Q

How can mutations outside of the coding region affect gene expression?

A

Mutations in promoter regions, where transcription factors bind, can deactivative a gene’s expression or make constantly active

Creation of an alternative splice site, competes with that of normal splice site in RNA processing

Results in proportion of mRNA with improperly spliced intron sequences

Can mutate out a splice site

155
Q

How can resistance develop in bacteria to anti biotics?

A

Stops a compound getting in, block entrance of a drug or pump it out

Modify target of a drug: eg a protein

Modify drug that comes in, new enzyme, for example B lactamase changes the B lactam ring of penicillin

Making more of the target, the drug can not cope

156
Q

What is the constitutive secretory pathway?

What is the regulated secretory pathway?

A

Constitutive: continuous and unregulated, packaged into vesicles and released by exocytosis.

EG: collagen, serum albumin, immunoglobulins

Regulated: released in response to a signal. Packaged into vesicles but not released until a stimulus is received by a hormone or neurotransmitter.

EG: insulin, glucagon, digestive juices

157
Q

Describe an overview of the secretory pathway in mammalian cells

A
  1. Free ribosomes initiates protein synthesis from mRNA molecule
  2. Hydrophobic N terminal signal sequence is produced
  3. Signal sequence of newly formed protein is recognised and bound to by the signal recognition particle SRP
  4. Protein synthesis stops
  5. GTP-bound SRP directs the ribosome synthesising the secretory protein to SRP receptors on the cytosolic face of the ER.
  6. SRP dissociates
  7. Protein synthesis continues and the newly formed polypeptide is fed into the ER via a pore in the membrane (peptide translocation complex)
  8. Signal sequence is removed by signal peptidase once the entire protein has been synthesised.
  9. The ribosome dissociates and is recycled.
158
Q

What protein modifications occur in the

ER?

Golgi?

A

ER:

signal cleavage by SIGNAL PEPTIDASE

disulphide bond formation PROTEIN DISULPHIDE ISOMERASE

N linked glycosyltion, oligosacchardies, asparagine side chains with amide group via DOLICHOL

GOLGI:

O linked glycosylation, GLYCOSYL TRANSFERASE, adds sugar to OH group, important in proteoglycans

Timming and modification of N linked oligosaccharides

Further proteolytic processing, exoproteases, endoproteases

Phosphorylation of monnose, to OH group, lysosome signal

159
Q

What occurs in N linked glycolsylation?

and O linked glycosylation?

A

N LINKED: in ER

Built up DOLICHOL PHOSPHATE carrier sitting in the membrane

Oligosaccharide transferred to amide group of ASPARAGINE

O LINKED:

-OH groups of SERINE and THREONINE

GLYCOSYL TRANSFERASE, builds up sugar chain from nucleotide sugar substrates

160
Q

Why is proteolytic procesing important in the formation of some secreted proteins?

A

ENDOPROTEASES/EXOPROTEASES

N terminal sequence removed in the ER, PRE SEGMENT

Further processing of PRO SEGMENT in the GOLGI

Examples

Preproalbumin

Preproinsulin

161
Q

How is insulin formed?

A

PreProInsulin translated, A, B and C peptides

Signal sequence cleaved by SIGNAL PEPTIDASE in the ER

ProInsulin has A, B and C peptides

Endopeptidases cleave the C peptide, in post golgi vesicle, so good measure of endogenous insulin

Insulin: A and B peptides joined by 2 disulphide bridges, made in ER

Active form

162
Q

Describe the structure of collagen

A

TROPOCOLLAGEN UNIT
Left handed triple helix of alpha chains

Primary sequence repeats GLYCINE-X-Y

Proline and hydroxyproline usually X and Y

Non compressable, high tensile strength, non extendable

Proline residues maintain correct shape, extended a helix

Hydroxyproline by PROLYLHYDROXYLASE, increase amount of H bonds, needs Vitamin C and Fe2+ for activity

Scurvy, vitamin D deficiency, weak tropocollagen helixes

163
Q

How is collagen synthesised?

CHASPOGRL

A

C leavage of signal peptide

H ydroxylation of proline/lysin

A ddition of N linked oligosaccharides to hydroxylysine

S ulphide(di) bond formation

P rocollagen

O linked glycosylation

G olgi to exocytosis

R emoval of N/C terminal peptides

L ateral aggregation to form fibrils

164
Q

How is tropocollagen cross linked?

A

Lysine residues to aldehyde derivatives

By LYSYL OXIDASE

Aldehydes then spontaneously form Aldol cross links

Needs vitamin B6 and Cu2+ for activity

165
Q

Describe how proteins are targeted to the nucleus

A

Through nuclear pores

Proteins contain a Nuclear Localisation Sequence, NLS

Run of 4 basic amino acids, internal

Folded

Importin recognises NLS, mediates transport inside

Importin binds to Ran GTP, back out

Ran back to nucleus with hydrolysis of GTP

166
Q

Describe how proteins are targeted to mitochondria

A

Transported unfolded

Signal sequence, N terminal, amphipathic

Unfolded proteins stabilised by chaperones like MSF (Mitochondrial Import Stimulating Factor)

Signal sequence recognised by TOM (Translocase Outer Membrane)

Those for matrix through TIM (Translocase Inner Membrane)

Needs ATP energy and a membrane potential

Signal cleaved by mitochondrial processing peptidase

167
Q

Describe how proteins are targeted to the lysosomes

A

Addition of mannose 6 phosphate to N linked oligosaccharides

N acetyl glucosamine phosphotransferase/phosphoglycosidase

Targeted for M6P addition by signal patch

M6P groups recognised by receptors in the trans Golgi

Vesicles pinched of for transport to lysosomes

Acid pH in lysosymes causes dissociation of the protein and receptor

Receptor back to golgi, phosphate removed from M6P group to ensure it does not travel back to Golgi

168
Q

What is I cell disease?

A

Genetic defects in N acetylglucosamine phosphotransferase enzyme

Lack of M6P addition

Lysosomal hydrolases mistargeted for secreted

Found in blood and urine

169
Q

How are proteins targeted for retention in the ER

A

Proteins such as disulphide isomerase and signal peptidase, resident in ER

Sometimes lost in vesicles pinched off to go to Golgi

Allows ER proteins to be retrieved

ER residents have a KDEL sequence (lys-asp-glu-leu)

C terinus

Interact wth KDEL receptors in golgi, enhanced by low pH

ER proteins bound to KDEL receptors return to ER in transport vesicles

Dissociate from KDEL receptros in neutral conditions of ER, KDEL back to golgi

170
Q

Describe the structure and mode of action of selected antibiotics and growth inhibitors

A

Bacterial cell wall is targeted by PENICLLIN, inhibits TRANSPEPTIDASE ENZYME that forms cross links in the cell wall, osmotic pressure causes cell lysis

Bacterial transcription targeted by RIFAMPCIN, binds to bacterial RNA polymerase preventing transcription

Bacterial protein synthesis targeted by TETRACYCLINE. Competes with tRNA at A site of bacterial ribosome

Anti folates in cancer therapy targeted by METHOTREXATE. Impairs the synthesis of TETRAHYDROFOLATE, which is essential for DNA synthesis, from folic acid. Competitively inhibits DIHYDROFOLATE REDUCTASE (DHFR)

171
Q

What is cloning?

A

The product of exact copies of DNA

172
Q

What is DNA sequencing?

A

the process of determining the precise order of nucleotides in a DNA molecule

173
Q

What is restriction analysis?

Describe how it is carried out

A

Restriction endonucleases (enzymes) are produced by bacteria

They recognise specfic DNA sequences, restrcition sites, and cut double stranded DNA

Restriction sites are often palindromic

Usually produces sticky ends which are staggered cuts

DNA ligase can be used to join the sugar phosphate backbone together

Used with electrophoresis

Investigates the size of the DNA fragements (deletions), investigate mutations, DNA variation (fingerprinting), gene cloning

174
Q

How are genes cloned using bacteria?

A

Plasmids used, small circular DNA that can transfer to other bacteria and contains antibiotic genes

Same restriction enzyme used to cut gene of interest and plasmid vector

Hydrogen bonds match sticky ends

DNA ligase joins the backbone

Recombinant DNA created

Introduced into bacterium = TRANSFORMATION

Bacteria with recombinant DNA identified, select for with ampicillin

Bacteria multiply

175
Q

Explain gel electrophoresis and how it is used to provide information about DNA fragments

A

Used to seperate DNA fragments of different sizes

Solution of fragments placed in well at negative electrode

DNA molecules are negatively charged - low pK

Travel towards positive electrode

Smaller molecules travel further

Agarose gel used

Buffer used to maintain charge on DNA samples across gel

Power supply generates charge difference

Stain: ethidium bromide fluoresces under UV light, added

176
Q

What is PCR?

A

Polymerase Chain Reaction

Amplified DNA segments by repeated copying of target DNA using a thermo stable DNA polymerase and a pair of DNA primers which uniquely define the region to be copied.

177
Q

What DNA polymerase is used in PCR?

What else does PCR require?

A

Thermostable

Taq DNA polymerase

from Thermus Aquaticus

Forward and reverse sequence specific primers, short oligonucleotides that can be 3’ extended

DNA nucleotides

178
Q

Why use PCR?

A

Amplify specificy DNA fragment

Specific, can discover novel sequences

Template DNA can be minute, crude sample from cheek swab

Investigate single base mutations e.g. Tay Sachs, SCD

Investigate small deletions or insertions e.g. CF

Investigate variation, genetic relationships

Diagnosis of inherited diseases

Detect presence of tumour cells

Early stages of infection

179
Q

Explain the process of PCR

A

Denaturation at 95C to spereate strands

Annealing of sequence specific primers to targt DNA, at 55C

Sample is heated to 72C to allow DNA synthesis by Taq polymerise, attaches free nucleotides

Exponential decrease

Extend 3’ end

180
Q

Describe SDS page

A

Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis

Seperate proteins on the basis of size only, molecular weight

Detergent, SDS, denatures protein molecules’ secondary and tertiary structure

Gives a negative charge proportional to Mr

One SDS per 2 amino acids

Visualised with Coomassie Blue dye

181
Q

Why is protein electrophoresis useful?

What does it required?

A

Needed to check protein levels, enzyme activity etc

Proteins are charged

Will move towards anode or cathode

Can be seperated on the basis of size, shape, charge

Required:

Gel, matrix to allow seperation

Buffer, to maintain protein charge

Power supply, to generate charge difference

Stain, Coomassie Blue

182
Q

Describe isoelectric focusing

A

Seperation of basis of charge, isoelectric point

pH gradient across gel

Protein migrates until pI = pH

No net charge at this point so migration stops

183
Q

Describe 2D page

A

Can seperate proteins with the same pI

But different molecular weights

Combines SDS PAGE and isoelectric focusing

Gel turned by 90 degrees and run for different property to seperate bands out

Seperate complex mixtures

Important for diagnosing disease states in different tissues, changes in smear intensity/size

Important technique for proteomics, protein analysis

184
Q

Describe proteomics

A

Analysis of all proteins expressed from a genome

Digest protein with trypsin

Mass spec

List of peptide sizes

Use database to identify

185
Q

What is an enzyme assay?

Describe

A

Measurement of enzyme activity in lab

Clinically useful to tell if an enzyme is present at normal levels

Performed at optimal pH, temperature and ionic strength

Appropriate ions and cofactors must be included

Production of product or disappearance of substrate is measured

Performed at high [substrate] = Vmax

Activity of enzymes in serum often measured, marker of tissue damage

If an enzyme from a particular tissue is found in serum it can indicate tissue damage caused by disease

186
Q

What are some examples of clinically important serum enzymes and what they mark for

A

Aspartate transaminase (AST) and alanine transaminase (ALT): markers for liver damage/disease

Creatine Kinase: marker for myocardial infarction

Amylase/lipase: markers for pancreatitis

y glutamyl transferase: marker for liver damage/ alcohol

Alkaline phosphatase: bone disorders

Acid phosphatase (ACP): Prostate cancer

Plasma cholinesterase: Decreased in liver disaease, inhibited in organophosphate poisoning

187
Q

What is western blotting and what is it used for?

A

After SDS PAGE

Seperated proteins traansferred to nitrocellulose membrace

Specific proteins can be visualised

Antibodies bind

Which are conjugated to an enzyme or fluorescent label

188
Q

What is ELISA?

What is it used for?

Describe

A

Enzyme Linked ImmunoabSorbant Assays

Concentration of a protein can be analysed in a complex mixture by binding of its corresponding antibody

Primary antibody is immobilised on a solid support

Solution to be assayed is applied to the surface

Antibody binds specific protein, others washed off

Second enzyme linked antibody binds to antibody-antigen complex

Binding of second antibody is measured by assaying the enzymes activity, rate at which it converts substrate to colour

189
Q

What are antibodies?

A

Bind to specific antigens by recognising a few specific amino acids

Monoclonal: 1 epitope, 1 antigen

Polyclonal: 1 antigen, many epitopes

Epitiope is the part of an antigen recognised by an antibody

190
Q

What is ELISA used for?

A

Insulin

Cortisol

Adrenaline

Noradrenaline

TSH

T3/T4

191
Q

What is Phaeochromocytoma?

A

Tumour mass secreting lots of adrenaline

192
Q

What is the sanger chain determination method?

Describe

A

Dideoxy chain determination

Produced in reads of 300 to 500 bases

Denature template, add labelled primer, DNA polymerase

Add radioactively, fluorescent ddNTPs which lack a OH on C3 as well as on C2

As well as dNTPs

This means no 3’ elongation by DNA polymerase

So the strand terminates

Depending on which ddNTP is used, the new strand will stop at different places

Range of new DNA fragments of different lengths, deantured with heat and seperated by gel electrophoresis

Add to polyacrylamide gel, each runs in a different lane and end result allows us to read the DNA sequence

193
Q

What is fluorescent dideoxynucleosidetriphosphate sequencing?

A

All in the same tube

Same lane

Different fluorescent marker for each base

Read by computers, graph

194
Q

How it DNA currently sequenced?

A

Fully automated

200 human genomes sequenced per week

Much cheaper

195
Q

Who would be interested in seeing an individuals genome information?

Ethics

A

Family

Potential spouses

Doctors

Governments

Police

Schools

Insurance companies

Who owns a genome?

196
Q

What is the importance of PCR?

A

Amplifies a small amount of DNA into many many copies, can be a crude smaple

Basis of DNA profiling

197
Q

What is reverse transcriptase PCR?

A

RNA is the demplate, instead of DNA

Prior to PCR, a cDNA is made by reverse transcriptase enzyme

Primer is complementary to the poly A tail

RNAse breaks down the RNA afterwards

Introns are left out

198
Q

What are other reasons for PCR?

(restriction)

A

Single base mutations can create a restriction site: JUNCTIONAL EPIDERMOLYSIS BULLOSA

Bg/II site created

PCR region around it

cut with Bg/II

Run on gel

Followed by restriction analysis, electrophoresis, DNA sequencing, Southern blotting, another PCR, nested, primers inside

199
Q

What is hybridisation?

A

The process of forming a DNA duplex between complimentary nucleic acid strands, normally DNA, for the detection of specific sequences.

Strands are often referred to as the probe and the target

200
Q

What is a probe?

A

A stretch of single stranded DNA

Labelled with radioactivity or fluorescence

Used in hybridiation studies

The presence or absence of a complementary stretch of nucleic acid can be established

201
Q

What is Southern Blotting?

A

DNA electrophoresis and hydbridisation

Digest with restriction enzymes

Electrophoresis

Transfer from gel to nitrocellulose/nylon, soak up liquid

Hybridised with labelled gene probe to show specific complimentary DNA fragments, in paper bag

Visualised by X ray

202
Q

Why is southern blotting used?

A

Investigate gene structure, large deletions or duplications

Investigate gene expansions, triplet repeats, fragile X, Hungtinton’s

Investigate variation, DNA fingerprinting

Investigate mutations in genetic tests, allele specific probes, SCD

203
Q

What is Northern blotting?

What is Western blotting?

A

Similar processes

N: RNA

W: proteins

204
Q

How can allele specific tests be carried out?

A

PCR: use primers specific for a sequence either side of an allele of interest to amplify. Primers for disease causing mutations. Only amplified if correct mutated primer. 3’ base of primer corresponsd with one allele or the other

Restriction analysis: use one or multiple restriction enzymes with sites in and aruond the allele. Analyse the size of fragments produced. If the restriction enzyme cuts the wild type but not the same, the site is mutated or missing

DNA/Southern Hybridisation: use allele specific oligonucleotides, for wild type and mutant.

205
Q

What is microarray?

Describe?

A

ARRAY COMPARITIVE GENOMIC HYBRIDISATION

ARRAY CGH

Screens for sub-microscopic chromosomal deletions for which the locus can not be deduced from the patient’s phenotype

Glass slides with thousands of dots to analyse thousands of genes, minute drops of liquid. Exact position of each gene known. Isolate mRNA, only expressed genes shown. No dot, unexpressed.

  1. An array of DNA probes covering the entire genome is applied to the surface of a solid matrix
  2. Patient DNA and normal control DNA are each labelled with different coloured fluorescent TAGs, eg patient red, normal green
  3. Equal amounts of labelled DNA are then hybridised to the probe array and the hybridisation signals are detected and compared
  4. For probes where the signal of normal DNA exceeds that of the patients DNA, more green than red, the patient has a deletion of the chromosomal region from which that probe was derived.

Computer compares levels of red and green

Look at deletions and duplications

206
Q

What tests can be used to analyse DNA at the gene level?

A

Southern blotting

R T - PCR

Microarray: Array CGH

207
Q

What is a karyotype?

A

Picture of a full set of stained METAPHASE chromosomes of an individual, ordered according to chromosome number

Different stains give different banding patterns

Determine sex, chromosome numbers and size

Organised in pairs

Chromosome 1 is the largest

23rd pair is X and Y

208
Q

What is FISH?

A

Fluorescent In Situ Hybridisation

Single stranded nucleic acids, usualyl DNA but can be RNA

Permitted to interact so that complexes are formed with sufficiently similar complementary sequences

Investigation of specific DNA sequences on chromosomes inside the cell

Label with fluoresence, can be for specific genes to locate, denature and hybridise

Chromosome painting, each a different colour

Can tell if large chunk are missing

Investigate genes in situ

Investigate chromosome number, structure and behaviour

209
Q

What is the relationship between changes in nucleotide and amino acid sequences?

A

Change in genetic code can result in different amino acids being coded for

EG AUA to AUG: Isoleucine to Methionine

A change in the primary sequence of a protein can lead to a change in the shape and therefore function of a protein

210
Q

What does a mutation =

A

Sequence variation

211
Q

What is a point mutation?

A

A change of a single nucleotide in a nucleic acid sequence

Base substitution

212
Q

What are the two types of base substitution?

Explain each

A

Transition:purines to purines, pyrimidines to pyrimidines

Transversion: purine to pyrimidines, pyrimidines to purines, less common

213
Q

What are the most common type of mutation?

How common are single base substitutions?

A

Single Nucleotide Polymorphisms: 2/3s of SNPs are C to T

Every 300 bp

214
Q

How many amino acids are there?

How many codons are there for each?

How many stop codons are there?

A

20 amino acids

1, 2, 3, 4 or 6 codons per amino acid

3 stop codons

215
Q

How can mutations in non coding regions or outside genes be detrimental?

A

Can alter

Binding sites

Promoter sequences

Splice sites

216
Q

What is the effect of a silent mutation?

A

One base substitution which does not change the amino acid in which the original codon coded for

Single base substitutions in the 3rd codon position do often not cause an amino acid change

217
Q

What is the effect of a missense mutation?

A

Replaces the original specified amino acid with another

Usually a single base change

218
Q

What codon positions do most non-silent mutations occur at?

A

1 and 2

219
Q

What is the effect of a nonsense codon?

A

Amino acid codon becomes a stop codon

Premature stop codon, PTC

220
Q

What is the effect of an insertion or deletion of 3 bases?

A

No reading frame change, but a change in the primary sequence, insertion of amino acid or deletion

221
Q

What is the effect of an insertion or deletion of 1 or 2 bases

A

Frameshift mutation

Reading frame of mRNA changed

Shifts triplet code along one or two nucleotides

Often causes a premature stop codon

222
Q

How can a silent mutation still cause disease?

A

Can alter splice sites

Might cause a new splice site within an exon

223
Q

How are PTCs protected against?

A

Nonsense mediated decay

Degrades PTCs

Little or no of the faulty protein produced

Protection from truncation

224
Q

What is the meaning of a conservative missense mutation?

A

Some amino acid substitutions are bettwer tolerated than others

Valine to alanine may be tolerated in non critical regions

Both hydrophobic

Evolved to be next to each other in the genetic table

225
Q

What is the effect of a mutation in:

Promoter region?

Start/stop codon?

Intron splice sites?

A

Promoter: transcription factors or RNA polymerase may njot bind

Start/stop: met-tRNA won’t bind, PTC, longer protein

Intron splice: skipping of adjacent exon, if exon 3bp multiple, short mRNA in frame. If not, mRNA short with frameshift, PTC and nonsense mediated decay

226
Q

How might spontaneous mutations occur?

A

Not caused by exposure to known mutagen

Errors in DNA replication

DNA bases have slight chemical instability

DNA strand slippage, new strand loops out, extra nucleotide. Template strand loops out, one less nucleotide

Tautomeric shift: proton briefly changes position, altered base properties, behave as different template base for DNA polymerase, C and A, T and G

Rate of spontaneous mutations depends on size and sequence

Usually in non coding regions

227
Q

How might an induced mutation occur?

A

Chemicals and radiation

Agents that cause mutations are mutagenic

Agents that cause cancer are carcinogenic

Examples: alkylating agents remove a base

Acridine agents add or remove a base

X rays break chromosomes, delete nucleotides

UV radiation creates thymidine dimers

Nitrous acid, changes base pairing

Ethidium bromide and IQ food mutagen intercalate to force bases apart

EMJ removes purine rings

228
Q

What is a mutation?

A

A change in a nucleic acid sequence

Which can be the addition of one or many nucleotides

The removal of one or many nucleotides

The rearrangement of several or many nucleotides

229
Q

Describe the most common effects of mutations

A

Not good or bad, just different

Source of genetic variation

Causes a mutant phenotype, differing from the wild type

Mutant allele

230
Q

What is a wild type?

A

An individual within a population displaying a wild type trait

The trait which is most common in the population

231
Q

What type of mutation can be passed onto the offspring?

A

Occurs in the germline

Gametes

232
Q

Describe the process of mismatch repair

A

Enzymes detect nucleotides that don’t base pair in newly replicated DNA

Incorrect base pair is excised and replaced

This is proof reading

233
Q

Describe the process of excision repair

A

DNA can accumulate damaged bases through oxidation, alkylation, deamination, uracil

Damaged DNA is removed by excision of bases

Replace a patch of DNA with DNA polymerase

Base excision repair: 1-5 bases, repairs oxidative damage, ROS

Nucleotide excision repair: from external agents, UV damage and carcinogens,, replaces up to 30 bases

234
Q

What do mutations in the DNA repair protein genes do?

A

Mutations accumulate

MASH1, 2, 6 genes

Can be inherited

Not efficiently repaired

235
Q

What is DNA double strand break repair?

A

When both DNA strands are broken, which can cause chromosomal rearrangements

236
Q

What is the function of the protein p53?

A

Monitors the repair of damaged DNA

if damage is too severe = apoptosis

237
Q

How is cancer caused generally?

A

DNA damaged to such a huge extent

That apoptosis - promoted by p53 - does not occur

OR

damage leads to uncontrolled growth

So cancerous cells are produced

238
Q

Describe tumour formation

A

Derived from individual abnormal cells

Arise from lack of normal growth control

Generated by a multipstep process

More likely to arise from cells undergoing frequent cell division

All of the cells in a tumour are of the same type

Behaviour depends on cell type

239
Q

What 6 new capabilites do tumour cells have?

A

Dividing independently of external growth signals

Ignore internal anti growth signals

Avoid apoptosis, p53

Divide indefinitely without aging

Stimulate sustained angiogenesis

Invade tissues to establish secondary tumours

240
Q

What is an oncogene?

A

Cancer causing gene

In control of cell division

241
Q

What is a protooncogene?

A

key amino acid substitutions can active into dominantly acting cancer causing oncogenes

242
Q

What is a tumour suppressing gene?

A

A gene involved in protecting the cell against one step on the path to cancer

243
Q

How is cancer heritable?

A

Inherited cancer genes, recessive mutations

Development, dominant inheritance pattern

Initiation of tuimour formation means that both copies are mutated or the functional copy is deleted

Mechanisms of homozygosity: loss of wild type chromosome, deletion of functional gene, point mutation, mitotic recombination

244
Q

How can viruses cause cancer?

A

Carry oncogenes

Presence of a virus means that the gene does not function as normal

245
Q

Why is PCR so important in the diagnosis of genetic disease?

A

Most mutations are single base changes so are hard to detect

PCR amplifies DNA segments by repeated copying using Taq polymerase, with primers that uniquely define the sequence to be copied

In sickle cell disease the restriction site for MstII is destroyed, so with gel electrophoresis and southern blotting there will be one less DNA fragment digested with MstII

246
Q

Describe MPCR

A

Multiplex PCR bases test for most common mutations of a gene

247
Q

What is MLPA?

A

Multiplication ligation dependent probe amplification

Exon counts for many exons duplicated in parallel

DNA denatured and mixed with MLPA probes, oligonucleotides containing one of the PCR primer sequences

If both probes hybridise they ligate and are PCRed

Osteogenesis imperfecta and duchenne muscular dystrophy

248
Q

How can DNA be sampled from an unborn child?

A

Parent DNA from blood or saliva

Foetal from:

Amniotic fluid cells, 12-20 weeks, guided by ultrasound, culture for 2 weeks, 0.5-1% chance of miscarriage

Chorionic villus biopsy, 10 - 13 weeks, transcervical, abdominal, 2% risk of miscarriage

foetal DNA is motehrs blood isolated

249
Q

What are some ethical issues associated with genetic testing?

A

Prantally used in abortion cases

Can screen for disease and prevent and treat harmful effects

Fetal screening, is ti a form of eugenics?

Neonatal can identify abnormalities to treat early

Carrier status, whethere or not to have a child if both carriers

Also children being tested for late onset dominant disease such as Huntington’s, would the older generations want to know?

Insurance access

Employers deciding who would suit jobs

Testing when no cure - psych issues

Selection for better children, messing with God’s creation?

250
Q

How does EM radiation cause mutations?

A

Short wavelengths, more damaging

Ionising radiation, produces ions when interacting with cellular moelcules

Solar radiation, X rays, nuclear power plant accidents, environmental sources, food, radon

UV light, all types damage collagen, skin aging

Sun burn and skin cancer from UVB

UV light photons cause adjacent thymine pases to pair, often resolved through spontaneous photo reactivation

251
Q

What is a chromosome?

How many in a diploid cell?

A

A nuclear thread like structure which carries the genetic information

Only visible when condensed in cell divisio

23 pairs

Each is one linear DNA molecule

252
Q

What are chromosomes made of?

A

CHROMATIN

which is made of:

DNA

Non-histone proteins

RNA

Histones: H1, H2A, H2B, H3, H4

H1 varies between species

H3 and H4 are highly conserved throughout evolution

253
Q

How is DNA arranged around histones?

A

Wrapped around an octamer of histones

H2A, H2B, H3 and H4 inside octamer

H1 stabilises

Histones are responsible for the beads on a string structure

Higher order structures are stabilised by hanging loops of DNA onto a protein scaffold, tightly folded chains

254
Q

What is the difference between euchromatin and heterochromatin?

A

Euchromatin is lightly packed and under active transcription usually. ACETYL GROUPS, nucleosomes, neads on a string

Heterochromatin is tightly packed chromatin, not being expressed. METHYL GROUPS, solenoids, 30nm fibres

255
Q

Describe the chromosomal basis of sex determination

A

Normal Female: 46, XX

Normal Male: 46, XY

256
Q

What are numerical chromosomal abnormalities ?

A

A number of chromosomes other than 46

Polyploidy

Aneuploidy

257
Q

What is polyploidy?

What is a cause of polyploidy?

A

A number of chromosomes that is a multiple of the haploid number

Common in plants, some animals

Observed in normal human muscle and liver

n=23, 2n=46, 3n=69, 4n=92

Polysperm is a cause, 2 to 3% of all pregnancies are triploid, most miscarry, others die shortly after birth.

Tetraploidy is rarer, usually found at prenatal diagnosis

258
Q

What is aneuploidy?

A

An abnormal number which is not a multiple of the haploid number

Monosomy: loss of one chromosome

Trisomy: gain of a chromosome, exists as a triplet

259
Q

What are the causes of aneuploidy?

A

Non-disjunction in meitotic cell division, missing or have both homologus chromosomes in one gamete

Non disjunction in mitosis, two populations of cells with different karyotypes, MOSAICISM

Anaphase lag, chromosomes left behind at cell division, because of defects in spindle function of attachment of chromosomes. Laggin chromosome may be lost completely in meiosis or mitosis

260
Q

What is a structural chromosomal abnormality?

What are the two broad types?

A

Physical changes to one or more of the chromosomes

Balanced: when the change does not cause any missing or extra genetic information

Unbalanced: when the changes cause missing or extra genetic information

261
Q

Describe Down Syndrome

A

47, XY+21

Trisomy 21

Non disjunction

Risk increases with maternal age

Eye problems, hypothyroidism, intellectual disability, congenital heart failure, hearing disorders

Can also arise from chromsomal translocation

262
Q

What other trisomies are there other than Downs?

A

Edwards: Trisomy 18, few survive beyound 15 days

Patau: Trisomy 21, multiple congenital abnormalities, most die within a year

263
Q

What is translocation?

A

the breakage and reformation of chromosomes such that DNA can be exchanged between chromosomes

Usually results in abnormal phenotypes

Recipricol: between 2 non homologous chromosomes

Breakpoints often lie between genes

Risk of passing one derivative chromosome to offspring, unbalanced, disease

264
Q

What is Robertsonian translocation?

How does it cause Downs?

A

P fragments are lost on a copy of CH 21 and Ch 14

This leads to a superchromosome 14:21, as centromeres are acrocentric, near one end

Carrier still has balanced genetic information as p arms are very short

Problesm depend on how chromosome lines up on metaphase plate.

If superchromosome is on opposite side to normal 14 and 21, no problems

If superchromosome is on same side as normal 14, and opposite to normal 21, trisomy 14, and monosomy 21. Both terminate

If superchromosome lines up one same side as normal 21, then trisomy 21 and downs. Other is monosamy 14, not viable

265
Q

What are interstitial and terminal deletions?

A

Internally is interstitial

Terminal is at end

By breakage

Always unbalanced

Associated phenotype

May be large enough to see by microscopy

May need FISH

266
Q

What is duplication?

A

Some genetic material is doubled within a chromosome

267
Q

What is an inversion?

A

No loss of genetic material

Rearrangement within the same chromosome

268
Q

What is a ring chromosome?

A

Loss of telomeres or ends of both asrm and formation of a ring

269
Q

What is an isochromosome?

A

Creation of two non identical chromosomes

One is a combination of the two short arms

The other is a combination of the two long arms

270
Q

What happens to inactivated X chromosomes?

A

They form Barr Bodies at the periphery of the nucleus

Condense

271
Q

What are some common sex chromosome aneuploidies?

A

Turners: 45, X. Problems as monosomal for PAR genes in Y chromosome

Triple X: 47, XXX. Learning diabilites, scoliosis, small head, tall

Klinefelters: 47 XXY, male.Reduced testosterone, increased breast tissue, language and reading impairment

XYY syndrome: 47 XYY. Phenotype essentially normal, slightly lower IQ, increased growth rate, normal testosterone

272
Q

What stain is used in karyotypes?

How is it used for location purposes?

A

GIEMSA

Banding pattern standardised as a set of ideograms

Dark and light bands are numbered

273
Q

What cells are used for karyotypes?

A

Cell culture may be required

Bone marrow 0 to 1 day

Blood 2 to 3 days

Solid tissue, skin

Amniotic fluid

Chorionic villus

7 to 21 days

274
Q

Which groups of chromosomes have satellites on them?

A

D: 13, 14, 15

G: 21, 22

275
Q

What are the types of chromosomes?

How are they grouped?

What are the p arms and the q arms?

A

Metacentric, acrocentric (D and G), sub metacentric

Grouped according to size similarity and centromere location

P arm is short petit arm

Q arm is long arm

276
Q

Outline reasons for referall of patients for karyotyping

A

Congenital abnormalities

Prenatal screening for downs at increased maternal age, family history of chromosomal abnormality, abnormal ultrasound scan

Birth defects, malformations, mental retardation

Abnormal sexual development, eg Kleinfelters

Infertility

Recurrent foetal loss

Acquired: leukaemia and related disorders

277
Q

What diagnosis offers of chromosomal abnormalities

A

Accurate diagnosis and prognosis of clinical problems in patients

Better management of affected patients, eg hormone therapy in sex disorders

Understanding of future reproductive risks

Prenatal diagnosis and possible termination of affected pregnancies

ESA 1 Revision (8 decks)

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