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MGD Flashcards

0
Q

What is solubility?

A

The extent to which a molecule can form hydrogen bonds

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1
Q

What are the 6 main differences between prokaryotic and eukaryotic cells?

A

Prokaryotes have no separate nucleus
Prokaryotes do not have membrane bound organelles
Prokaryotes have a peptidoglycan cell wall
Prokaryotes lack most organelles (don’t have Golgi, lysosomes, RER, SER, mitochondria)
Prokaryotes have 70S ribosomes whereas eukaryotes have 80S
Prokaryotes have pili and flagellae

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2
Q

What does hydrophilic mean?

A

Water soluble
Polar
Can form hydrogen bonds and dissolve

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3
Q

What does hydrophobic mean?

A

Not water soluble
Non polar
Cannot form hydrogen bonds and can’t dissolve

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4
Q

What does amphipathic mean?

A

Molecule has hydrophilic/polar and hydrophobic/non polar regions

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5
Q

What is a Micelle?

A

Structure formed by amphipathic molecules-
Hydrophobic regions cluster together away from the water
Hydrophilic regions interact with water forming an ordered shell around the hydrophobic regions

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6
Q

How are biological membranes arranged?

A 
Phospholipid (amphipathic) bilayer
Intrinsic and extrinsic proteins embedded in the layer
Fluid mosaic model
Phosphorus head is hydrophilic
Fatty acid chains are hydrophobic
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7
Q

What is pH?

A

Measure of the H+ concentration of a solution

-log(10) [H+]

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8
Q

What is pKa/b?

A

The likeliness of an acid or base to dissociate in solution

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9
Q

The higher the pKa…?

A

The weaker the tendency of the acid to dissociate- weaker the acid (higher pH)

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10
Q

The lower the pKa…?

A

The stronger the tendency of the acid to dissociate- the stronger the acid (lower pH)

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11
Q

What is the Henderson Hasselbach equation?

A

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

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12
Q

Define a buffer solution

A

Mixture of a weak acid and its conjugate base which resist changes in pH when small amounts of acids or bases are added or diluted

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13
Q

If the R group of an amino acid is positively charged, is the amino acid considered acidic or basic?

A

Basic (as it is acting as a proton acceptor)

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14
Q

If the R group of an amino acid is negatively charged, is the amino acid considered acidic or basic?

A

Acidic (as it is acting as a proton donor)

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15
Q

What is the value of pK when the amino acid is acting as a base?

A

pK > 7

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16
Q

What is the value of pK when the amino acid is acting as an acid?

A

pK < 7

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17
Q

If the pH of the surrounding solution < pK of aa

Then is the aa protonated or deprotonated?

A

Protonated (aa acting as a base)

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18
Q

If the pH of the surrounding solution > pK of aa

Then is the aa protonated or deprotonated?

A

Deprotonated (aa acting as an acid)

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19
Q

Define the isoelectric point

A

pH at which the protein has no overall net charge

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20
Q

What is the pI value of basic proteins and what does this infer about the amino acids in the protein?

A

pI>7

Most amino acids are basic/ positively charged

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21
Q

What is the pI value of acidic proteins and what does this infer about the amino acids in the protein?

A

pI< 7

Most amino acids are acidic/ negatively charged

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22
Q

If the pH of the surrounding solution < pI of the protein

Then is the protein mostly protonated or deprotonated?

A

Mostly protonated

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23
Q

If the pH of the surrounding solution > pI of the protein

Then is the protein mostly protonated or deprotonated?

A

Mostly deprotonated

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24
Q

Name 5 important uses of proteins

A 
Catalysts
Transporters
Structural support
Immune protection
Ion channels
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25
Q

How do amino acids exist in the body?

A

As zwitterions

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26
Q

What is a peptide bond?

A

Bond formed in a condensation reaction between two amino acids
C(=O)–N(-H)

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27
Q

What are three features of a peptide bond?

A

Planar- all elements lie in one plane
Rigid (double bond characteristics)- no rotation about double bond; delocalised electrons = shorter and more stable bond
Trans orientation - of carboxyl oxygen and amide hydrogen

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28
Q

How can amino acids be classified?

A

Aliphatic/ Aromatic
Polar/ non polar
Charge of R groups

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29
Q

What 9 amino acids are non polar?

A 
Glycine
Alanine
Valine
Leucine
Isoleucine
Methionine
Proline
Phenylalanine
Tryptophan
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30
Q

What 6 amino acids are polar and uncharged?

A 
Serine
Threonine
Tyrosine
Cysteine
Glutamine
Asparagine
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31
Q

What 3 amino acids are polar and positively charged?

A

Histidine, lysine, arginine

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32
Q

What 2 amino acids are polar and negatively charged?

A

Glutamate, aspartate

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33
Q

What 2 amino acids are helix breakers?

A

Proline and glycine

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34
Q

What two amino acids are helix formers?

A

Alanine and leucine

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35
Q

What is interesting about the size of glycine?

A

Very small R group

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36
Q

Why is proline a good helix breaker?

A

Peptide bond is in a cyclic arrangement and so rotation of bonds either side of the peptide bond is IMPOSSIBLE
So groups can’t move to form bonds which would form the helical structure

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37
Q

What is the primary structure of proteins?

A

The amino acid sequence of a protein.

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38
Q

What is the secondary structure of proteins?

A

Stretches of the polypeptide chain that form a-helices and b-sheets
Bonds on either side of the peptide bond can rotate freely. When these angles remain the same throughout a segment of polypeptide the protein adopts a regular secondary structure

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39
Q

What is the tertiary structure of proteins?

A

The full 3D structure of the protein. Involves the folding up of the secondary structures. Improper folding (Amyloidoses) may cause disease.
Most proteins fold spontaneously, but some require the help of molecular chaperones.

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40
Q

What is the quarternary structure of proteins?

A

Interaction between and arrangement of different polypeptide chains (subunits) within the same protein. The polypeptide chains may be identical (homomeric) or different (heteromeric).

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41
Q

What is the structure of alpha helices?

A

3.6 amino acids per turn
0.54 nm pitch
Right handed helix
C=O group of one amino acid is H bound to N–H group of a residue 4 amino acids away
H bonds are roughly parallel to axis of helix
R groups found on outside of alpha helix

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42
Q

What is the structure of B strands?

A 
Extended conformation
0.35nm between adjacent amino acids
R groups alternate
Can form anti parallel, parallel or mixed sheets 
Multiple inter strand H bonds
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43
Q

How does protein folding occur?

A

Spontaneously or with the help of chaperones

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44
Q

What is amyloidosis?

A

Improper folding of polypeptide sequence (tertiary structure of protein) which may cause disease

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45
Q

What is the structure of amyloid fibres?

A

Misfolded protein
Highly ordered B sheet
H bonds between B sheet cause protein aggregation

Interchain assembly of B strands are stabilised by hydrophobic interactions between aromatic amino acids (aromatic amino acids block the interaction of amino acid side chains- and stops the aggregations of fibrils)

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46
Q

What is the structure of fibrous proteins?

A

One repeating secondary structure
Little/ no tertiary structure
Long strands/ sheets
Usually insoluble

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47
Q

What is the structure of globular proteins?

A

Several types of secondary structure
Complex tertiary structure
Compact
Usually soluble

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48
Q

What is the role of fibrous proteins?

A

Structure
Support
Protection

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49
Q

What is the role of globular proteins?

A

Enzymes

Regulatory proteins

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50
Q

What bonds are found in the primary structure of proteins?

A

Covalent (peptide)

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51
Q

What bonds are found in the secondary structure of proteins?

A

Hydrogen bonds

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52
Q

What bonds are found in the tertiary and quarternary structure of proteins?

A 
Covalent (disulphide)
Hydrophobic interactions
Ionic interactions
Hydrogen bonds
VDW
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53
Q

What is the structure of haem?

A

Complex of protoporphyrin IX and Fe2+

Fe2+ bound to 4 nitrogens (ring structure), and bound to histidine residue on globin chain

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54
Q

Where is myoglobin found?

A

Heart and skeletal muscle

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55
Q

What is the function of myoglobin?

A

Acts as an oxygen reservoir in heart and skeletal muscle
Oxygen carrier
Increases rate of transport of oxygen in muscle cell

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56
Q

What is the structure of myoglobin?

A

1 polypeptide of 153 amino acids
Compact
75% a- helical
His93 residue is in the 8th a- helix covalently bound to Fe2+
Hydrophobic centre and hydrophilic surface

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57
Q

What happens to a molecule of myoglobin when oxygen binds?

A

Hyperbolic dependence
Fe 2+ in deoxygenated myoglobin is slightly below the plane of the protoporphyrin ring
Oxygen binding causes the movement of Fe2+ into the plane of the ring
Causing the movement of Histidine F8 and a change in the protein conformation

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58
Q

Describe the shape of the myoglobin oxygen binding curve

A

Rectangular hyperbole

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59
Q

Do the laws of km and vmax apply to myoglobin?

A

Yes

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60
Q

Does myoglobin show cooperativity?

A

No

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61
Q

Where is haemoglobin found?

A

Exclusively in RBCs

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62
Q

What is the structure and function of haemoglobin?

A 
2 a (141 aa) and 2 b (146 aa) subunits = heterotetramer
Each polypeptide is associated with haem
Non covalent interactions hold the 4 chains together

Carry oxygen from lungs to tissue

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63
Q

How can the structure of haemoglobin be determined?

A

Through crystallisation and a series of X ray shots

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64
Q

Why is the binding curve of haemoglobin different to that of myoglobin when they have very similar structures?

A

Mb had a very high affinity for oxygen and so will only release oxygen when pO2 is very low
Hb however can exist in 2 states- a low affinity T state and a high affinity R state. The transition between these two states gives Hb its sigmoidal binding curve - Hb’s affinity for oxygen increases as more oxygen binds- cooperativity

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65
Q

What is the T state of haemoglobin?

A

Tense state
Low affinity of oxygen
Right
T state
Stabilised by low pH, high co2 and high BPG
When positive histidine residue is attracted to the negative aspartate residue

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66
Q

What is the R state of haemoglobin?

A

Relaxed
High affinity for oxygen
Left
More stable

Stabilised by higher pH low co2 and low BPG

When histidine residue moves down- so there is no more interaction between histidine and aspartate

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67
Q

What is cooperative binding?

A

Binding of one molecule promotes the binding of another molecule

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68
Q

Does haemoglobin show cooperativity?

A

Yes

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69
Q

What is the shape of the haemoglobin oxygen binding curve?

A

Sigmoidal

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70
Q

What is the effect of 2,3-BPG on Hb?

A

Decreases the affinity of Hb for O2
1 binds per tetramer
Favouring the T state
Curve shifts to the right

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71
Q

What is the effect of BPG at high altitudes on Hb?

A

BPG conc increases at high altitudes which lowers Hb’s affinity for oxygen - promoting the release of oxygen into tissues
R converts to T state

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72
Q

What is the effect on BPG during metabolism on Hb?

A

Large amounts of metabolism produces large amounts of BPG which lowers Hb’s affinity for oxygen- so O2 is released more readily in areas performing large amounts of metabolism

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73
Q

What is the effect of carbon dioxide and H+ on Hb?

A 
High carbon dioxide and H+ (highly metabolic tissues) 
Decreases the affinity of Hb for oxygen
Curve shifts to the right R to T state
H+ protonates His residue 
Carbon dioxide binds at N terminus
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74
Q

What is the effect of carbon monoxide on Hb?

A

Binds to Hb 250x more readily than oxygen
Decreases affinity of Hb for oxygen
Fatal when COHb > 50%

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75
Q

What effect does the binding of CO to one Hb have on the binding of oxygen to other residues?

A

Binding of CO acts to increase the affinity of unaffected sub units for oxygen

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76
Q

What is the difference between adult Hb and foetal Hb and whi is this difference important?

A

HbF = 2alpha and 2gamma
HbA = 2alpha and 2beta
HbF has a higher affinity for oxygen than HbA
Ensures oxygen can be obtained from mother

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77
Q

In sickle cell anaemia what base change occurs?

A

A to T

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78
Q

In sickle cell anaemia what amino acid change occurs?

A

Glutamate to valine

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79
Q

What is the pathophysiology of sickle cell anaemia?

A

Glutamate is a polar negative amino acid
Valine is a non polar neutral amino acid
In the T state= Formation of hydrophobic pocket as a result of polymerisation of haemoglobin molecules due to valine residues
This results in a distortion of the RBCs to a sickle shape

Sickle Hb = 6-8g.dl-1
Normal Hb = 14g.dl-1

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80
Q

What is alpha thalassaemia?

A

Decreased or absent alpha chains
Beta chains can form stable tetramer a with a higher affinity

Caused by 2 genes on chromosome 16 - varying levels of severity
Onset before birth

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81
Q

What is beta thalassaemia?

A

Decreased or absent beta chains
Lack of beta chains causes alpha chains to precipitate out of solution or form tetramers with gamma chains

Chromosome 11- major and minor
Symptoms appear after birth

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82
Q

How do enzymes work?

A

Enzymes work by lowering the activation energy needed for a reaction to occur. Binding of substrate to a distinct part of the enzyme, the active site, increases the local concentration of reactants and also stabilises the formation of the high energy transition state

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83
Q

How does temperature affect the rate of a reaction?

A

Increases the number of molecules with energy greater than the activation energy and hence rate of reacton

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84
Q

How does the concentration of substrate affect the rate of the reaction?

A

Increases the chance of molecular collisions and hence the rate of reaction

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85
Q

What is the active site?

A

Cleft or crevice in the globular structure of an enzyme where the substrate weakly binds to form the enzyme substrate complex
It is formed by only a few amino acids from different parts of the primary sequence
It excludes water - preventing hydrolysis from occurring and interference of reaction

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86
Q

Do enzymes affect the position of an equilibrium?

A

No they just increase the rate of attainment of equilibrium

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87
Q

What is the lock and key hypothesis?

A

Substrate has a complementary shape to the active site and binds to it without changing its shape

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88
Q

What is the induced ft model?

A

Active site only forms a complementary shape after binding to the substrate as the active site slightly changes shape to fit around the substrate

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89
Q

What is the michaelis menten equation?

A

Vo= Vmax [S] / km + [S]

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90
Q

What curve does the michaelis menten give and what does this tell us about the enzymes it can be used for?

A

Rectangular hyperbolic

Simple Enzymes which can only exist in one conformation

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91
Q

What is vmax?

A

The maximal rate of reaction when all enzyme active sites are saturated with substrate

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92
Q

What is km?

A

Substrate concentration that gives half the maximal velocity

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93
Q

What is the significance of km?

A

Gives a measure of the affinity of an enzyme for its substrate

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94
Q

What does a low km suggest?

A

High affinity of enzyme for substrate

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95
Q

What does a high km suggest?

A

Low affinity of enzyme for substrate

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96
Q

What is the significance of the lineweaver burk plot?

A

X intercept = -1/km

Y intercept = 1/vmax

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97
Q

What do irreversible inhibitors do to enzymes?

A

Bind covalently to the active site

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98
Q

What do reversible competitive inhibitors do to enzymes?

A

Binds at the active site temporarily
Affect km
Does not affect vmax

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99
Q

What do reversible non competitive inhibitors do to enzymes?

A

Binds at a secondary site, alters enzyme conformation
Affects vmax
Does not affect km

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100
Q

How are enzymes regulated in the short term?

A

Changing substrate and product concentration

Changing the conformation of the enzyme- allosteric, covalent modification, proteolytic cleavage

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101
Q

How are enzymes regulated in the long term?

A

Change in rate of protein synthesis

Change in rate of protein degradation

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102
Q

What is substrate concentration regulation?

A

Availability of substrate affects rate of enzyme activity

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103
Q

What is product concentration regulation?

A

Accumulation of product inhibits the forwards reaction

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104
Q

What is an example of product concentration regulation?

A

Accumulation of G6P in step 1 of glycolysis inhibits hexokinase activity

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105
Q

What is allosteric regulation?

A

With allosteric affectors- which bind to a site other that the active site, and change the enzyme conformation, changing the activity of the enzyme by stabilising the R(highaff) or T(lowaff)state of the enzyme
Enzymes have more than one sub unit and exist in 2 states (high affinity R state and low affinity T state)
Sigmoidal relationship between [S] and rate (so km and vmax do not apply)
The binding of substrate to one subunit makes subsequent binding to other subunits progressively easier- positive cooperativity
These allosteric effectors may either inhibit or activate an enzyme

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106
Q

What do allosteric activators do?

A

Bind to site other than active site
Alter the conformation of the enzyme
Increase the proportion of the R state enzymes

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107
Q

What do allosteric inhibitors do?

A

Bind to site other than active site
Alter the conformation of the enzyme
Increase the proportion of the T state enzymes

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108
Q

What is an example of allosteric regulation?

A

Phospho fructokinase

Step 3 of gycolysis key regulatory step

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109
Q

What activates phospho fructokinase?

A

AMP, fructose 2 6 BISPHOSPHATE

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110
Q

What inhibits phospho fructokinase?

A

ATP citrate H+

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111
Q

What is covalent modification?

A

Many different types of group can be attached covalently to proteins (in this case enzymes) via amino acids. Most importantly, phosphate groups can be added (phosphorylation) [Ser,Thr OH]. The attachment of a phosphate group is carried out by kinase enzymes and their removal by phosphatase enzymes (regulated by hormonal levels)

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112
Q

What enzymes attach a phosphate group to a molecule?

A

Kinase

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113
Q

What enzymes remove a phosphate group from a molecule?

A

Phosphatase

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114
Q

Discuss the concept of enzyme cascades and the use of protein kinases and phosphatases to regulate activity

A

When enzymes activate other enzymes, the number of affected molecules increases in the enzyme cascade.

Kinases transfer the phosphate group from ATP to the –OH group of Ser, Thr, Tyr
Phosphatases remove phosphate groups through hydrolytic activity.

Phosphate groups are bulky, charged groups that can significantly affect enzyme conformation and substrate binding. The addition and removal of these groups therefore regulates enzyme activity.

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115
Q

What is proteolytic cleavage?

A

Enzyme secreted as an inactive protein precursor (zymogen) and cleaved by proteases to the active enzyme.

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116
Q

What is a zymogen?

A

Inactive precursor of an enzyme

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117
Q

What is an example of proteolytic cleavage?

A

Blood clotting cascade

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118
Q

What enzyme is secreted in the stomach? What is its inactive and active form? What activates it?

A

Inactive: Pepsinogen
Active: Pepsin
Activated by pH

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119
Q

What enzyme is secreted in the pancreas (T)? What is its inactive and active form? What activates it?

A

Inactive: Trypsinogen
Active: Trypsin
Activated by enteropeptidase

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120
Q

What enzyme is secreted in the pancreas (CT)? What is its inactive and active form? What activates it?

A

Inactive: chymotrypsinogen
Active: chymotrypsin
Activated by trypsin

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121
Q

What enzyme is secreted in the pancreas (CP)? What is its inactive and active form? What activates it?

A

Inactive: procarboxypeptidase
Active: carboxypeptidase
Activated by trypsin

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122
Q

What enzyme is secreted in the pancreas (E)? What is its inactive and active form? What activates it?

A

Inactive: proelastase
Active: elastase
Activates bye trypsin

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123
Q

What is the blood clotting cascade?

A

Example of proteolytic cleavage
Coagulation (intrinsic and extrinsic pathway) + common pathway
Formation of a fibrin clot through a series of proteolytic cleavages

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124
Q

What is the intrinsic pathway of the bcc?

A

Endothelium lining tear
Endothelial cells produce von willebrands factor
VW factor activates platelets and stimulates their adhesion to exposed laminin and collagen
Activates factor 12
Activates factor 11
Activates factor 9
Activates factor 10 with help of factor 8
Common pathway

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125
Q

What is the extrinsic pathway of the bcc?

A 
Endothelium lining tear
Endothelial cells produce thromboplastin (tissue factor)
Thromboplastin activates factor 7
Activates factor 10 
Common pathway
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126
Q

Describe how a fibrin clot is formed/ common pathway of bcc?

A

Activated Factor 10 activates prothrombin to thrombin with the help of factor 5
Thrombin activates fibrinogen to fibrin
Thrombin also activates factor 13
Factor 13 helps fibrin become stabilised fibrin= fibrin clot

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127
Q

How is the blood clotting process stopped/ regulated?

A

By the removal of activated proteins (dilution of factors by bloodstream to liver)
Proteolytic digestion (protein c activated by thrombin binding to thrombodulin- digests factors 5 and 8
Binding of inhibitor molecules (antithrombin 3 acts on unbound thrombin - enhanced by heparin binding)
Fibrinolysis (tPa and streptokinase activates plasminogen to plasmin; plasmin activates breakdown of fibrin clot to fibrin fragments)

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128
Q

How is a fibrin plot broken down?

A

Fibrinolysis- tPa and streptokinase activates plasminogen to plasmin; plasmin activates breakdown of fibrin clot to fibrin fragments

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129
Q

What is the importance of vitamin k in the blood clotting cascade?

A

Vitamin K is required to produce inactive factors in the liver
Forms gla residues that targets factors to the calcium in the membranes using carboxylase enzyme

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130
Q

What is the importance of calcium in the blood clotting cascade?

A

Calcium accumulates in the endothelial lining
Positive charge attracts the negatively charged gla residues on the inactivated factors bringing them to the site of damage
Calcium required to assist with the activation of factors 2,7,9,10 (same as those that require vit K in their synthesis)

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131
Q

What is the importance of gla residues in the blood clotting cascade?

A

Synthesised by vit k on factors
Negatively charged
Attracted to calcium in endothelial lining- activated

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132
Q

How does warfarin work as an anticoagulant? When is it used?

A

Inhibits the vitamin K dependent factors in the intrinsic and extrinsic pathways 2thrombin, 7, 9, 10
Prevents the formation of gla residues on the factors
So factors not targeted to endothelial lining and calcium
So factors not activated

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133
Q

What is the relevance of the use of the drug streptokinase in the bcc?

A

Used to activated conversion of plasminogen to plasmin

Hence breakdown of a fibrin clot by plasmin action

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134
Q

Describe the positive feedback involved in the blood clotting cascade

A

Activation of thrombin promotes further activation (of factors 8,5,13 assistant factors)

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135
Q

Why is positive feedback important in the bcc?

A

Because only a small amount of initial factor is required to produce a big response of clotting

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136
Q

What does a nucleotide consist of?

A

Phosphate
Pentose sugar
Base

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137
Q

What does a nucleoside consist of?

A

Pentose sugar and base

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138
Q

What is the significance of nucleosides?

A

Can cross cell membranes more easily than nucleotides (without the large and negative phosphate)

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139
Q

What is the structure of a ribose sugar? Where is it found?

A

OH at carbon 2

Found in RNA

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140
Q

What is the structure of deoxyribose sugar? Where is it found?

A

H at carbon 2

Found in DNA

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141
Q

What are purines?

A

Double ring structured bases

A, G

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142
Q

What are pyrimidines?

A

Single ring structured bases

C, U, T

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143
Q

How do bases pair up?

A

Purine with a pyrimidine
A with T (2 H bonds)
C with G (3 H bonds)

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144
Q

What is the structure of RNA?

A

Single stranded
A C G U
Forms a stem loop structure- hydrogen bonds are formed e tween anti parallel complementary sequences on the same strand of RNA (single strand loops back on itself do that one side will run anti parallel and H bonds will form between complementary bases)

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145
Q

What is the structure of DNA?

A

Double stranded helix
A C T G
Two polynucleotides are completely complementary and anti parallel
10 BP per turn
3.4nm per turn (between turns)
0.34 nm between base pairs
Stabilised by van der waals forces above and below the ring
Have major (exposed bases) and minor grooves (which are not at 180 degrees from one another)

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146
Q

How are DNA and RNA represented by hand?

A

Both DNA and RNA are labelled 5’ to 3’ (5’ starts with phosphate)
Top strand is 5’ to 3’
Various bases are given letters, eg A, T, G, C, U
Duplex structure includes the complimentary antiparallel strand
Hydrogen bonds are denoted by dotted lines

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147
Q

What is the polarity DNA/ RNA?

A

Nucleotides are covalently linked via phosphodiester bonds.
Each single-strand nucleic acid chain has a polarity.
Two distinct ends – 5’ end with free phosphate and a 3’ end with free –OH

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148
Q

How is eukaryotic DNA condensed into a nucleosome and solenoid structure?

A

Nucleosomes – DNA is wound (~ twice) round histone core, which is charged
Each nucleosomes is coiled to form solenoid structures
Solenoid structures are further condensed into chromatids

Shcjhebihcbuiwdbciubwdoucbouwbecuwed chromo abno

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149
Q

What is heterochromatin?

A

Condensed DNA which is not being expressed / transcribed/ cannot replicate
Appears darker
(Mitosis chromosomes- ie are involved in cell division)

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150
Q

What is euchromatin?

A

Uncondensed DNA which is being expressed / transcribed/ can replicate
Appears lighter
(Interphase chromosomes)

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151
Q

How many chromosomes do humans have?

A

46

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152
Q

How many chromatids does a replicating cell have?

A

92

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153
Q

How many chromosomes does a gamete have?

A

23 chromosomes

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154
Q

What does mitosis produce?

A

2 identical diploid cells

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155
Q

What does meiosis produce?

A

4 non identical haploid cells

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156
Q

What is the cell cycle?

A

Interphase -
G1- cell content replication
S- DNA replication
G2- cell check and quick repair before division

Mitosis- cell division

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157
Q

Where are the cell cycle check points found?

A

At the end of G1

At the end of G2

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158
Q

What is the importance of the cell cycle check points?

A

Important for regulation

In cancer cell cycle no longer functions so there is continuous growth of cells

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159
Q

What is the relevance of G0 and where is it found?

A

Comes off of G1

Importance icuhbefhu bweifcjniedcedcjinedc?

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160
Q

What catalyses DNA replication?

A

DNA polymerase

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161
Q

What tools are required for DNA replication?

A 
Primate
Helicase
DNA polymerase- required activated precursors dNTPs and ATP since every addition to chain requires hydrolysis of ATP 
Ligase
DNA nucleotides
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162
Q

In what direction does DNA polymerase create the complementary DNA strand in replication?

A

5’ to 3’

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163
Q

What is the initiation process for DNA replication?

A

DNA helicase unravels the DNA double helix
Primase binds to the origin of replication recruiting DNA polymerase to the site (DNA polymerase can only add bases in a 5’ to 3’ direction)

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164
Q

What is the elongation process for DNA replication?

A

Leading strand is replicated in a 5’ to 3’ direction continuous, as normal
Lagging strand is replicated discontinuously in Okazaki fragments (in 5’-3’ direction too)
[Since DNA polymerase can only extend strands in the 3’ direction only one of the two new strands can be synthesised in a continuous manner in the same direction in which the replication fork is moving]

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165
Q

What is the termination process for DNA replication?

A

Replication forks move from the ends of the DNA strands towards each other and meet in the middle
[Lead strands move towards lagging strands and vice versa]
Okazaki fragments are then joined by DNA ligase from OH group to Phosphate group covalently

166
Q

Why is DNA replication known as semi conservative replication?

A

Semi conservative in that after replication each chromosome now consists of one strand of original DNA and one new strand

167
Q

What is mitosis important for?

A

Growth
Repair
Maintenance

168
Q

What are the 6 stages of mitosis?

A 
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
Cytokinesis
169
Q

In mitosis what happens in prophase?

A

Nuclear membrane disappears
Chromosomes condense (heterochromatin)
Spindle fibres appear

170
Q

In mitosis what happens in prometaphase?

A

Spindle fibres attach to the centromeres of chromosomes

Chromosomes continue to condense

171
Q

In mitosis what happens in metaphase?

A

Chromosomes line up randomly in single file along the metaphase plate (centre of cell)

172
Q

In mitosis what happens in anaphase?

A

Spindle fibres contract
Centromeres divide
Sister chromatids move too opposite poles

173
Q

In mitosis what happens in telophase?

A

Nuclear membrane reforms
Chromosomes decondense
Spindle fibres disappear

174
Q

In mitosis what happens in cytokinesis?

A

Cytoplasm divides

Parent cell becomes 2 daughter cells with identical genetic info

175
Q

Why is meiosis important?

A

Maintains diploid chromosome number in zygote

176
Q

What are the stages of meiosis?

A

Prophase - cytokinesis 1
Prophase - cytokinesis 2

(Effectively two rounds of mitosis)

177
Q

How is meiosis different to mitosis? 3 ways?

A

Meiosis produces 4 non identical gametes, whereas e mitosis produces 2 identical daughter cells
In meiosis there are two cell divisions whereas there is only one in mitosis
In meiosis, homologous pairs are first divided and then sister chromatids, whereas in mitosis sister chromatids are divided straight away

178
Q

How does meiosis bring about variation?

A

Independent assortment of chromosomes at meiosis

Crossing over

179
Q

What is independent assortment of chromosomes?

A

Independent assortment is the random distribution of maternal and paternal chromosomes into gametes during meiosis.

180
Q

What is crossing over?

A

In bivalent/tetrad forms of chromosomes- bits of DNA swap from one homologous chromosome to another- increasing genetic variation

181
Q

Define a genotype

A

Genetic make up of an organism either as a whole or for a specific genetic locus

182
Q

Define a phenotype

A

All observable characteristics of an individual or the observable trait as the result of the genetic make up of the one or more specific genetic loci

183
Q

How does the environment affect genotype and phenotype?

A 
Radiation
Mutagens
Chemicals that affect cell growth
Diet 
Lifestyle
184
Q

What is co dominance and an example?

A

Where two alleles of a gene have equal dominance and both affect the phenotype equally
Blood type

185
Q

What is complementation and an example?

A

When more than one gene is involved in the expression of a genotype- means that parents with different affected alleles can have unaffected children
Albinism

186
Q

What is linkage?

A

Genes close to one another on the same chromosome are said to be linked
Genes on different chromosomes or far away from one another on the same chromosome are said to be not linked
Linked genes do not show independent assortment at meiosis (as they co segregate and are inherited together)

187
Q

What types of genes do not show independent assortment at meiosis?

A

Linked genes

188
Q

What is recombination frequency?

A

The likelihood of crossing over occurring between two linked genes

189
Q

If you have a high recombination frequency (50%) what does this suggest?

A

High likelihood of crossing over occurring between two genes
Genes are unlinked
RF=50% because when material splits during meiosis the 2 genes will either end up on the same chromatid or will separate (2 options with equal chance)

190
Q

If you have a low recombination frequency (<50%) what does this suggest?

A

Low likelihood of crossing over occurring between two genes

Genes are linked

191
Q

How do you calculate recombination frequency?

A

Total number of confirmed recombinant progeny/ total number of confirmed progeny
Confirmed- we are certain of their genotype

192
Q

What is a gene?

A

Gene - A unit of heredity; a length of DNA on a chromosome that contains the code for a protein

193
Q

What is an allele?

A

Allele - An alternative form of a gene; each individual has two alleles for every gene, which can either be the same or different.

194
Q

What is dominant?

A

Dominance - A phenotypic trait is dominant when it occurs in both homo and heterozygotes.

195
Q

What is recessive?

A

Recessive – A phenotypic trait is recessive when it occurs only in homozygotes

196
Q

What is autosomal?

A

When the gene in question is located on an autosome

197
Q

What is X linked inheritance?

A

When the gene in question is located on a sex chromosome

198
Q

What is a heterozygous genotype?

A

Two alleles are different

199
Q

What is a homozygous genotype?

A

Two alleles are the same

200
Q

What is a hemizygous genotype?

A

When there is only one allele instead of two for a gene in a diploid organism
Normal- sex chromosomes
Abnormal- missing autosomal chromosomes

201
Q

What is autosomal dominant and an example?

A

When individuals have a heterozygous or homozygous dominant (fatal) genotype for a disease on their autosomes
Males ~ females
Cannot skip a generation
Affected individuals have a 75% chance of having affected offspring

Hunting tons
Marfan’s

202
Q

What is autosomal recessive and an example?

A

When individuals have a homozygous recessive genotype for a disease on their autosomes
Heterozygotes are carriers
Males ~ females
2 homozygous recessive individuals will only have affected children
2 carriers will have 25% chance of having affected offspring (parents don’t need to be affected for child to be)
Can skip generations

Cystic fibrosis, albinism

203
Q

What is x linked recessive and an example?

A

When individuals have a homozygous recessive genotype (women) or a hemizygous recessive genotype (men) on their sex chromosomes
Males > females
Heterozygous female has a 50% chance of having affected sons
Affected males cannot give their allele to their sons (only give Y chromosome)
Can skip generations
Girls can inherit defective allele from mum and dad, boys can only inherited defective alleles from mother
Haemophilia A
Duchenne muscular dystrophy

204
Q

What are ribosomes?

A

Complex of rRNA and many proteins that act as a mini protein factory
Makes proteins in process of translation by reading RNA template

205
Q

What is mRNA?

A

2% of all RNA
1000s of different kinds but few copies of each
Single strand with no stem loops, have codons
Made by RNA polymerase II

206
Q

What is tRNA?

A

15% of all RNA
100s of different kinds, many copies of each
Single strand with stem loops, have anticodons
Made by RNA polymerase III

207
Q

What is rRNA?

A

80% of all RNA
4 kinds in eukaryotes but many copies of each
Associated with proteins to form ribosomal subunits
Made by RNA polymerase I

208
Q

How does protein synthesis differ in prokaryotes?

A 
Bacteria have:
Simpler promoter regions
Different transcription factors
Coupled transcription and translation
No PTM so mRNA is short lived
70S ribosomes
Different transcription factors and initiation mechanisms
209
Q

What is the process of initiation in transcription?

A

Transcription factor binds to Promoter sequence TATA box 30 bases upstream of origin of transcription
Recruits RNA polymerase II - separates DNA to allow RNA nucleotides to bind and forms phosphodiester bonds

210
Q

What is the process of Elongation in transcription?

A

RNA polymerase II binds to template strand (3’-5’) and makes pre mRNA in 5’-3’ direction (copies coding strand)

211
Q

What is the process of termination in transcription?

A

RNA polymerase II recognises a stop codon and falls off

212
Q

What are the three main post transcriptional modification that occur to pre mRNA to form mRNA?

A

Post transcriptional modification
5’ capping- methylated guanine added to 5’ end
3’ Polyadenylation- lots of A’s added to 3’ end
PREVENT DEGRADATION
Splicing- introns removed by endo/exonucleases from pre mRNA = mRNA (only contains exons)

213
Q

What is the process of initiation in translation?

A

40S ribosome subunit with methionyl tRNA binds to AUG start codon on mRNA.. Binding causes 60S subunit to combine with the 40S subunit initiating translation to occur

Can be free in the cytoplasm or bound to ER

214
Q

What is the process of elongation in translation?

A

Met tRNA occupies the P site of the ribosome
Another anticodon is recognised, so next amino acyl tRNA occupies the A site
Peptidyl tranferases catalyse the formation of peptide bond between the amino acids in the P and A site
tRNA in P site becomes uncharged and so is released- both amino acids now occupy the A site
Ribosome shifts one to the right- moving the dipeptide into the P site and freeing up that A site for the addition of another amino acyl tRNA
Continues

215
Q

What is the process of termination in translation?

A

Stop codon is recognised on mRNA
No tRNA with complementary anticodons exist so polypeptide is hydrolysed from tRNA using H20 to release it into the cytoplasm

216
Q

Where does transcription occur?

A

Nucleus

217
Q

Where does translation occur?

A

Cytoplasm

218
Q

What does degenerative mean?

A

There is more than one triplet code coding for one amino acid

219
Q

Where are proteins, synthesised in ribosomes attached to ER, destined to go?

A

Membrane or secretory pathway

220
Q

Where are proteins, synthesised in ribosomes free in the cytosol, destined to go?

A

Cytosol or post translational import into the cell

221
Q

How do ribosomes attach to the ER?

A

Via co translational insertion

222
Q

What 4 things are required for protein sorting?

A

Signal, receptor, translocational machinery, Energy

223
Q

Describe the generic secretory pathway

A

Ribosome begins to synthesise a polypeptide chain from mRNA
The polypeptide chain will have a hydrophobic signal at it’s N terminus
This signal is recognised by a signal recognition particle which binds to the signal, stopping translation temporarily, and the SRP directs the ribosome to the ER
At the ER the SRP binds to a receptor which temporarily fixes the ribosome to the ER
SRP dissociates and this restarts translation
The polypeptide sequence enters the ER via a membrane pore, whilst being synthesised
If protein is for secretory pathway signal peptide is cleaved within the ER
If protein is for the membrane it will have another signal peptide which anchors it in the membrane and continues translation on the cytosolic side of ER

224
Q

What is the SRP?

A

Multi domain riboprotein which mediates a 3 way association with SRP receptor in the ER, the ribosome and signal peptide

225
Q

What is the nature of the signal that targets proteins to the ER?

A

Hydrophobic N terminus signal sequence
13-36 amino acids long
1+ positively charged residues & 10-15 hydrophobic residues at N terminus
A few hydrophilic residues within C terminal region

226
Q

Is the protein folded or unfolded in the transfer into the ER?

A

Unfolded

227
Q

What specialist proteins are involved in the targeting of proteins to the ER?

A

SRP, SRP receptor, protein translocator

228
Q

Is the signal retained or cleaved on a protein being targeted to the ER?

A

Cleaved by signal peptidase

229
Q

Does the targeting of a protein to the ER require energy?

A

Yes- hydrolysis of GTP by SRP

230
Q

What is the nature of the signal that targets proteins to the nucleus?

A

NLS= nuclear localising signal
Basic (Arg and lys residues)
May be multipartite
Various positions, must be on the surface of the folded protein

231
Q

Is the protein folded or unfolded in the transfer into the nucleus?

A

Folded (can pass through the large pores in the double membrane of the nucleus)

232
Q

What specialist proteins are involved in the targeting of proteins to the nucleus?

A

Importin - recognises NLS and mediates transport to nucleus

RanGTP displaces importin in nucleus and drives out export cargo

233
Q

Is the signal retained or cleaved on a protein being targeted to the nucleus?

A

Signal is retained - to facilitate the reimporting of proteins when nucleus reforms after cell division

234
Q

Does the targeting of a protein to the nucleus require energy?

A

Yes - the hydrolysis of GTP

235
Q

What is the nature of the signal that targets proteins to the mitochondrial matrix?

A

Amphipathic signal for the initial targeting to matrix (may be extra signals to other final destinations) at the N terminus

236
Q

Is the protein folded or unfolded in the transfer into the mitochondrial matrix?

A

Held partially unfolded by chaperones (mitochondrial import stimulation factor)

237
Q

What specialist proteins are involved in the targeting of proteins to the mitochondrial matrix?

A

TOM (Translocase of the outer membrane)- recognise proteins on outer membrane and form import channels
TIM (Translocase of inner membrane)- proteins transported across the inner mitochondrial membrane

Chaperones of the HSP70 family- assist in folding

238
Q

Is the signal retained or cleaved on a protein being targeted to the mitochondrial matrix?

A

Cleaved by mitochondrial processing peptidase

239
Q

Does the targeting of a protein to the mitochondrial matrix require energy?

A

Yes- ATP hydrolysis by mitochondrial HSP70, drives translocation into the lumen and keeps some proteins unfolded prior to delivery.
Electrical potential across IMM assists translocation

240
Q

What is the nature of the signal that targets proteins to the lysosomes?

A

Post translational addition of mannose 6 phosphate to N linked oligosaccharides in the Golgi ( using N-acetylglucosamine phosphotransferase and N-acetylglucosamine phosphoglycosidase)
Proteins destined for lysosomes are targeted for the addition of M6P groups by the presence of a signal patch, a sequence of several amino acids from different parts of the amino acid sequence

241
Q

Is the protein folded or unfolded in the transfer into the lysosomes?

A

Folded (delivered via vesicles)

242
Q

What specialist proteins are involved in the targeting of proteins to the lysosomes?

A

M6P receptor in the trans Golgi

243
Q

Is the signal retained or cleaved on a protein being targeted to the lysosomes?

A

Phosphate group is removed from the M6P group on the protein by a phosphatase- to ensure that the protein does not return to the Golgi with the receptor

244
Q

Does the targeting of a protein to the lysosomes require energy?

A

Yes- phospho transferase

UTPNAG + mannose –> UMP + NAG + mannose-6-P

245
Q

What is the nature of the signal that retains proteins in the ER?

A 
KDEL signal (Lys-Asp-Glu-Leu)
At the C terminus
246
Q

Is the protein folded or unfolded in the retention of a protein in the ER?

A

Folded (transported as a vesicle)

247
Q

What specialist proteins are involved in retaining proteins in the ER?

A

KDEL receptor in the cis Golgi - protein binds at low pH

248
Q

Is the signal retained or cleaved on a protein being retained in the ER?

A

Retained

249
Q

Does the retention of a protein in the ER require energy?

A

Not directly- involves binding and release dependent on pH
Golgi- low pH
ER- neutral pH
But formation of budding vesicle requires GTP hydrolysis

250
Q

What are 7 functions of the ER?

A 
Insertion of proteins into membranes
Specific proteolytic cleavage 
N linked glycosylation 
Formation of disulphide bonds
Proper folding of proteins 
Assembly of multi sub unit proteins
Hydroxylation of proline and lysine residues
251
Q

What 3 main modifications occur in the ER?

A

Signal proteolytic cleavage
Disulphide bond formation
N linked glycosylation

252
Q

What 3 main modifications occur in the Golgi?

A

O linked glycosylation
Trimming and modification of N linked oligosaccharides (ie. addition of M6P for targeting to lysosomes)
Further proteolytic cleavage of some proteins- e.g activating an enzyme

253
Q

What proteolytic cleavage occurs in the ER?

A

Signal sequences are cleaved off (removal of pre sequence)

Signal peptidase

254
Q

What proteolytic cleavage occurs in the Golgi?

A

In collagen synthesis when 150 N terminal aa and 250 C terminal aa (pro peptide sequences which don’t form the triple helix) are cleaved off
(Removal of pro sequence)

255
Q

What glycosylation occurs in the ER?

A

N linked glycosylation
Oligosaccharides are built up on a dolichol phosphate carrier molecule sitting in the membrane
Oligosaccharides is then transferred to the amide group of asparagine
Oligosaccharide protein transferase

256
Q

What glycosylation occurs in the Golgi?

A

O linked glycosylation
Attachment of oligosaccharides to the hydroxyl group of serine and threonine
Glycosyl transferase builds up sugar chain from substrates
(Important in proteoglycans)

257
Q

Why are disulphide bonds formed in proteins in the ER?

A

They increase the stability of the protein in environments that can be harsher than those inside the cell
Protein disulphide isomerase

258
Q

What two proteins are resident ER proteins?

A

Protein disulphide isomerase

Signal peptidase

259
Q

Name 3 ER chaperones that try to help with folding problems

A

Binding immunological protein

Calnexin and calreticullin

260
Q

How can misfolding occur?

A

As a result of a mutation, causing the protein to be incorrectly associated with other proteins

261
Q

How does Binding immunological protein attempt to correct folding problems?

A

Binds to exposed amino acid sequences that would normally be buried in the interior of the folded protein
Retain folded protein in the ER
Act as sensors to monitor the extent of misfolding
Mediate increased transcription of chaperones and reduction in translation

262
Q

How does calnexin and calreticullin attempt to correct folding problems?

A

Bind to oligosaccharides on incompletely folded proteins
Retain folded protein in the ER
Act as sensors to monitor the extent of misfolding
Mediate increased transcription of chaperones and reduction in translation

263
Q

What happens if misfolding cannot be corrected by chaperones?

A

Protein may be returned to the cytosol for degradation

Protein may accumulate to toxic levels in the ER resulting in disease

264
Q

What is constitutive secretion?

A

Continuous process
Proteins packaged into vesicles and released continuously by exocytosis.
E.g. Serum albumin, collagen

265
Q

What is regulated secretion?

A

Proteins released in response to a signal e.g. hormone

Proteins packaged into vesicles but not released until stimulus received E.g. insulin

266
Q

What is the structure of collagen?

A

The basic unit of Collagen is Tropocollagen
Primary sequence is (Glycine-X-Y)n
X,Y= proline (helix breaker-prevents individual chains from forming helices), hydroxyproline (hydroxylated proline using prolyl hydroxylase, increases amount of interchain H bonds), lysine and hyroxylysine
3 tropocollagen alpha chains arranged in a triple helix structure (Left-handed triple helix – Non-extensible/compressible, high tensile strength)

o Prolyl Hydroxylase requires Vitamin C and Fe2+ ions for activity.
- Scurvy is due to low Vitamin C, therefore weak tropocollagen triple helices.

267
Q

What does prolyl hydroxylase do and what does it require to function?

A

Prolyl hydroxylase assists in the hydroxylation of proline residues in collagen
Requires vitamin C and Fe2+ for normal activity

268
Q

How is collagen synthesised? CHAD FATERLCA

A

Cleavage of signal peptide
Hydroxylation of proline and lysine residues
Addition of N linked oligosaccharides and galactose to hydroxylysine residues
Disulphide bond formation at C terminal propeptide sequence to hold alpha chains in place for helix formation
Formation of triple helical pro collagen from N terminal to C terminal
Addition of O linked oligosaccharides
Transport vesicle to cell membrane
Exocytosis at membrane into EC space
Removal of the N and C terminal propeptide sequences
Lateral association of collagen molecule
Covalent cross linking of collagen molecule
Aggregations of fibrils

269
Q

What is the structure of insulin?

A

Contains A and B peptide joined via 2 disulphide bonds

270
Q

What is the clinical relevance of the C peptide?

A

C peptide is a good marker for measuring the levels of endogenous insulin being produced in diabetic patients
Since insulin and C peptide are produced in the same amounts

271
Q

How is insulin synthesised?

A

PreProInsulin – Contains Signal Sequence, A, B and C peptides.
Signal Sequence is cleaved by signal peptidase and 2 disulphide bonds are formed between A and B proteins
ProInsulin – Contains A, B and C peptides
Endopeptidases cleave C peptide

272
Q

What does DNA sequencing do?

A

Determines the sequence of nucleotide bases in a DNA fragment

273
Q

What is the process of DNA sequencing?

A

SANGER method
· Uses modified ‘terminator’ nucleotides (ddNTP which lacks 3’ OH and thus stops elongation)
· 4 test tubes are set up each containing a single stranded DNA fragment (with an unknown sequence), free nucleotide bases, ‘terminator’ nucleotide bases (ddNTP, where N is A, T, C, G), a radioactively or fluorescently labelled primer, DNA polymerase
· Depending on which ddNTP is used and where the ddNTP binds to the DNA template (in 5’- 3’ direction), the synthesis of the new strand will stop at different places and thus varying lengths of DNA are synthesised
· All the fragments of new DNA in each of the test tubes will end with a nucleotide that has the same base
· DNA Gel electrophoresis: Samples are then loaded onto a polyacrylamide gel and the DNA strands separate according to their length
· Fragments can be seen on the gel because of their labelled primers

MODERN method
Fluorescent dideoxyDNA sequencing - the samples of DNA are run on one lane
· Each different ddNTP is labelled a different colour
· Cheaper and faster methods

274
Q

Where is DNA sequencing used?

A

· Determine the length and sequence of DNA
· Forensics
· Cloning

275
Q

What are restriction endonucleases and what do they do?

A

Bacterial enzymes that are able to recognise specific DNA sequences and then cut the double stranded DNA at that specific site

276
Q

What is the process of using restriction endonucleases?

A

· Specific restriction endonucleases recognise and cut specific DNA sequences by breaking the phosphodiester bonds
· Restriction endonucleases cut at palindromic sites
· Produce staggered ends- ‘sticky ends’ between which H bonds can form, but not phosphodiester bonds (can only be formed in the presence of DNA ligase)
· Restriction endonucleases also methylate the sticky ends of the DNA fragment to prevent other DNA fragments from rebinding

277
Q

Where can restriction endonucleases be used?

A

Locate specific sequences of DNA

Comparing number and location of restriction sites on two DNA strands- wild type and mutant type

278
Q

What is the process of restriction analysis?

A

Use of restriction endonucleases and them gel electrophoresis

279
Q

Where can restriction analysis be used?

A

· Investigate the size of the DNA fragments (identify deletions)
· Investigate mutations (sickle cell disease)
· Investigate DNA variation (DNA fingerprinting)
· Gene cloning (IN VITRO= PCR)

280
Q

What is the purpose of gene cloning?

A

To amplify the quantity of target DNA

Cam be done in vivo or in vitro

281
Q

What is the process of in vivo gene cloning?

A

· Using plasmids
o Small circular DNA
Can be transferred to other bacteria
o Can contain antibiotic genes
· Plasmid is cut using restriction endonucleases- gene is added to create a Recombinant DNA molecule
· Recombinant DNA is introduced into bacteria in transformation
· Antibacterial genes are used to positively select for bacteria that have taken up any plasmid (e.g. will now contain gene for ampicillin resistance so will survive when ampicillin is added), and bacteria which have taken up the recombinant DNA (e.g. recombinant DNA would have been added into the tetracycline gene; so replica plating occurs, and then tetracycline is added; cells containing recombinant DNA will die)
· Bacteria containing recombinant DNA are placed in an environment to multiply

282
Q

What does DNA gel electrophoresis do?

A

Separates strands of DNA according to size

283
Q

What is the process of DNA gel electrophoresis?

A

· Gel: Agerose
· Buffer: Allows charge on DNA samples across the gel
· Power supply: generates charge difference across the gel
· Stain/ detection: Ethidium bromide
· DNA fragments of known size are used as a reference

· DNA molecules are loaded into wells on a gel made of agerose (which has small holes in it and thus acts like a sieve)
· An electric field is supplied across the gel
· The negatively charged DNA (due to negative phosphate group) moves towards the anode (from negative electrode to positive electrode)
· Shorter DNA fragments travel further along the gel as longer DNA fragments get trapped in the small holes of the agerose gel more easily
· DNA fragments are stained with ethidium bromide (fluorescent dye) so that they can be seen on the gel

284
Q

Where can DNA gel electrophoresis be used?

A

· Size of DNA fragments

· Used with most other methods of analysis when identifying a gene

285
Q

What is the process of in vitro gene cloning / polymer chain reaction?

A

· Separation of DNA fragments: DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler; temperature is increased to 95 degrees causing the two strands of DNA to separate
· Addition (annealing) of primer: mixture is cooled to about 55 degrees (ranges from 55- 65; 65 is most common for G and C bases due to there being 3 H bonds being formed as opposed to 2 H bonds with A and T) causing the forwards and reverse primers to anneal to the complementary bases at the ends of the DNA fragment; Primers provide the starting sequence for DNA polymerase to begin DNA copying because DNA polymerase can only work in the 5’ to 3’ direction and attach at the end of an existing chain; Primers also prevent renaturation
· Synthesis of DNA: temperature is increased to 72 degrees (optimum temperature for DNA polymerase to add complementary nucleotides along each of the separated DNA strands); It begins at the primer on both strands and adds nucleotides in sequence until it reaches the end of the chain.

286
Q

What is the process of reverse transcriptase PCR?

A

Reverse Transcriptase-PCR
Form of PCR where mRNA is converted into cDNA using reverse transcriptase
Poly A tail on mRNA acts as a primer, enabling reverse transcriptase to convert the mRNA into cDNA
The cDNA is then amplified in PCR

287
Q

Where can PCR be used?

A

· Amplify specific DNA fragments
· Genetic tests -with the addition of a restriction endonuclease, we can identify conditions where a restriction site is created (such as Junctional Epidermolysis Bullosa) and where a restriction site is destroyed (Tay Sachs Disease)
· Single base mutations- Tay Sachs, Sickle Cell disease
· Investigating small deletions or insertions (Cystic Fibrosis)
· Investigating variation, genetic relationships (DNA profiling)

288
Q

Where can RT PCR be used?

A

RT PCR-
· Gene expression (if gene is being expressed then mRNA will be present)
· Produces DNA for PCR
· Can be used for genes from bacteria/viruses

289
Q

What does hybridisation/ blotting do?

A

Investigation of one gene on a DNA strand using a DNA probe

290
Q

What is southern blotting?

A

A technique where DNA separated by electrophoresis is transferred to a membrane filter and is detected by the hybridisation of a labelled probe

291
Q

What is the process of southern blotting?

A

· Extraction- DNA is extracted from samples and increased using PCR
· Digestion- DNA is cut into fragments using restriction endonucleases
· Separation- DNA fragments are separated according to size using gel electrophoresis (agerose gel); DNA fragments are immersed in alkali which causes the DNA to denature into single strands
· Blotting- DNA fragments are transferred from gel to nylon membrane by southern blotting- wodge of paper towels placed on top which assists the DNA being drawn up into the nylon; DNA fragments are fixed to the nylon with UV light
· Hybridisation- DNA probes are added to label the fragments by binding to specific fragments (complementary nucleotide base sequence to the gene we are interested in potentially finding)
· Development- membrane with labelled DNA fragments, is placed onto an X ray film; development of film shows dark bands where radioactive DNA probes have bound

292
Q

What is northern blotting?

A

A technique where RNA, separated by electrophoresis is transferred to a membrane filter and is detected by the hybridisation of a labelled probe

293
Q

Where can hybridisation/ blotting be used?

A

· Investigating gene structure- large deletions/ duplications
· Investigating gene expansions and triplet repeats- (E.g. Fragile X syndrome, Huntington’s)
· Investigating mutations in genetic tests- using allele specific probes (E.g with Sickle cell disease AàT)
· To investigate variation and genetic relationships- DNA fingerprinting

294
Q

What does hybridisation/ microarray / array CGH do?

A

Investigation of 1000’s of genes simultaneously
on a DNA strand using a DNA probe

(can also be used with RNA- reverse transcriptase= cDNA)

295
Q

What is the process of hybridisation/ microarray/ array CGH?

A

· DNA fragments organised into arrays are hybridised to labelled DNA from 2 different sources
· 2 different sources are labelled either red (e.g cancerous) or green (e.g healthy)
· Red and green fluorescence can be studied to work out the cancerous: healthy ratio for each cell (red: green)

  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 e.g. Patient’s DNA labelled red and normal DNA labelled green
  3. Equal amounts of the labelled DNA are then hybridised to the probe array and the hybridisation signals are detected and compared.
  4. For probes where the signal of the normal DNA exceeds that of the patient’s DNA, the patient has a deletion of the chromosomal region from which that probe was derived.
    (If Green > Red on the probe array solid matrix
296
Q

Where can hybridisation/ micro array/ array CGH be used?

A

· Investigate deletions/ duplications
· Investigate conditional gene expression- (Comparing cancerous and normal genes; Comparing patient and normal genes)
· Used to screen for sub microscopic chromosomal deletions for which location (locus) cannot be deduced from the patients phenotype

297
Q

What does karyotyping do?

A

Looks collectively at all chromosomes

298
Q

What is a karyotype?

A

A karyotype is a picture of the full set of stained metaphase chromosomes of an individual organised in pairs according to chromosome number

299
Q

How are chromosomes arranged in a karyotype?

A

· Chromosome 1 is the largest chromosome and chromosome 22 is the smallest
· Chromosomes are grouped and numbered according to their size and position of their centromere

300
Q

Where can karyotypes be used?

A

· Investigating chromosome deletions/ duplications
· Reasons for karyotyping include: Constitutional (congenital) abnormalities (prenatal screening, birth defects, abnormal sexual development, infertility, recurrent foetal loss) AND Acquired abnormalities (Leukaemia and other related disorders)

301
Q

What does FISH do?

A

Used to identify the presence and location of a region of DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections

302
Q

What is the process of FISH?

A

· Fluorescent probes for a specific gene (or several genes) can be used or probes for specific DNA stretches on chromosomes such as telomeres or centromeres
· For chromosome investigation chromosome painting is often used whereby each chromosome is visualised using a different coloured fluorescent probe

Denature the chromosomes
Denature the probe
Hybridization
Fluorescence staining
Examine slides or store in the dark
303
Q

Where can FISH be used?

A

· WITHIN cells
· Locating a specific gene on a chromosome
· Identifies chromosome abnormalities
· Degree of sequence identity can be determined

304
Q

What does protein electrophoresis do?

A

Separates protein fragments on the basis of molecular weight (size), shape or charge

305
Q

What 3 methods of protein electrophoresis are there?

A

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE)- Separation on basis of size/ molecular weight

Isoelectric Focusing- Separation on basis of Isoelectric Point/ Charge

2D polyacrylamide gel electrophoresis (2D-PAGE)- Separation of proteins with complex structure

306
Q

What is the process of SDS PAGE?

A

Protein molecules are denatured by B-ME (breaks disulphide bonds)and SDS (eliminates secondary/ tertiary structure) [One molecule of SDS binds for every 2 amino acids]
· The bound SDS has a large negative charge which masks the intrinsic charge of the protein
· Therefore protein fragments are separated due to molecular weight/ size

307
Q

What is the process of isoelectric focusing?

A

containing a pH gradient
· Protein will migrate in the electric field until it reaches a pH that matches it’s pI (Isoelectric point- protein has no overall charge at this point)

308
Q

What is the process of 2D PAGE?

A

· Combination of SDS PAGE and Isoelectric focusing

· Allows separation of proteins that have identical pI values but different molecular weights

309
Q

Where can protein electrophoresis be used?

A

Where amino acid deletions have occurred

Nonsense mutations have resulted in a PTC and a truncated protein

310
Q

What is the general process of Protein electrophoresis?

A

· Gel: Polyacrylamide
· Buffer: maintains charge on protein samples
· Power supply: generates charge difference across the gel
· Stain/ detection: coomassie blue

311
Q

What is western blotting?

A

a technique where a protein, separated by electrophoresis is transferred to a membrane filter and is detected by the hybridisation of a labelled probe

312
Q

What does Enzyme Linked Immuno Absorbent Assays (ELISA) do?

A

Determines whether a particular protein is present in a sample
Measures the concentration of proteins in solution (a complex mixture e.g. serum) by analysing the binding of antibodies

313
Q

What is the process of ELISA?

A

· Coating- Polythyrene plate is treated with a solution of either antigen or antibody
· Blocking- an unrelated protein based solution is used to cover all unbound sites on the plate
· Detection- Enzyme conjugated antibody or antigen binds specifically to the target antigen or antibody
· Read Results- Substrate is added and the signal (fluorescence) produced by the enzyme substrate reaction is measured

314
Q

What two things could the antibodies in ELISA be?

A

Antibodies used in ELISA’s can either be monoclonal or polyclonal

315
Q

What are monoclonal antibodies?

A

Antibodies derived from unique antibody producing cells called hybridomas and capable of specific binding to a single unique epitope

316
Q

What are polyclonal antibodies?

A

A pool of antibodies purified from animal sera that are capable of binding to multiple epitope

317
Q

Where can ELISA be used?

A

· Elisa tests are used to detect proteins (substances with antigenic properties)- hormones, bacterial antigens and antibodies

318
Q

What do enzyme assays do?

A

Measures the activity of an enzyme

Gives an indication of its presence at normal levels

319
Q

What is the process of enzyme assay?

A

· Measures the production of product/ disappearance of substrate
· Performed at optimal pH, temperature and ionic strength ( with ions and cofactors)
· Performed with a high concentration of substrate so that the enzyme works optimally
· Continuous (where the assay gives a continuous reading of activity)- E.g. spectrophotometry or chemiluminescence
· Discontinuous (where samples are taken, the reaction stopped and then the concentration of substrates/products determined)- E.g. radioactivity or chromatography

320
Q

Where can enzyme assays be used?

A

Important CLINICALLY-

The activity of various enzymes in serum is an indicator of tissue damage

321
Q

What is Aspartate transaminase/ alanine transaminase a clinical marker for?

A

Liver damage

322
Q

What is amylase/ lipase a clinical marker for?

A

Pancreatitis

323
Q

What is gamma glutamic transferase a clinical marker for?

A

Liver damage due to alcohol

324
Q

What is alkaline phosphatase a clinical marker for?

A

Bone dosorder

325
Q

What is acid phosphatase a clinical marker for?

A

Prostate cancer

326
Q

What is plasma cholinesterase a clinical marker for?

A

Decreased in liver disease

327
Q

What are creatine kinase and lactate dehydrogenase (and now more commonly troponins) a clinical marker for?

A

Myocardial infarction

328
Q

What is a mutation?

A

Mutation – ‘a change in a nucleic acid sequence, which can be the addition of one or more (or many) nucleotides [insertion], the removal of one or more (or many) nucleotides [deletions], or the rearrangement of several (or many) nucleotides’

329
Q

What is a point mutation?

A

A single base substitution

330
Q

What is a transition?

A

Substitution of a purine for another purine

Substitution of a pyrimidine for another pyrimidine

331
Q

What is a transversion?

A

Substitution of a pyrimidine to a purine or vice versa

332
Q

What is silent mutation?

A

A mutation that does not alter the amino acid specified

333
Q

What is a nonsense mutation?

A

A mutation that changes the amino acid specified to a stop codon
Results in a premature termination codon (PTC)
mRNAs containing PTCs are degraded by nonsense mediated decay (NMD) and little or no protein is produced

334
Q

What is a missense mutation?

A

A mutation that replaces one amino acid with another

335
Q

What is a frame shift mutation?

A

Addition or subtraction of nucleotides not in multiples of 3

336
Q

What 3 mutations can cause a frame shift mutation?

A

Deletion of nucleotides not in multiples of 3
Insertion of nucleotides not in multiples of 3
Intron splice site mutation - since exon after mutated site is deleted- if its not a multiple of 3 = frame shift

337
Q

What is a start codon mutation?

A

Mutation to a start codon which results in the protein no longer being transcribed or translated as it is not recognised

338
Q

What is a promoter sequence mutation?

A

Mutation to nucleotides in the promoter sequence where transcription factors bind
Activates / deactivates the promoter region
Alters transcription and gene expression

339
Q

How can an exon deletion or duplication mutation be detected?

A

By doing exon counts with MLPA

340
Q

What is a mutation to a stop codon?

A

Results in a delayed stop codon

Elongated non functional protein formed

341
Q

What are the spontaneous causes of mutations?

A

DNA replication- tautomeric shift and slippage

342
Q

What is tautomeric shift?

A 
Spontaneous cause of mutation
Altered base pairing due to a shift of a proton 
Bases act as other bases
C--> A
T--> G
343
Q

What is slippage?

A

Spontaneous cause of mutation
Newly synthesised strand loops out = extra base in new strand

Template strand loops out = loss of base in new strand

344
Q

What are the induced causes of mutations?

A

Mutagens- nitrous acid, ethyl methane sulphonate, iQ/ ethidium bromide
Ionising radiation - UV

345
Q

How does nitrous acid cause mutations?

A

C –> U binds with A
A –> H binds with C
G –> X binds with C

346
Q

How does ethyl methane sulphonate cause mutations?

A

Removal of purine rings

Any base can pair with a purinic site

347
Q

How does IQ/ ethidium bromide cause mutations?

A

Disrupts DNA packing
Intercalation of IQ/ EB forces bases further apart
Leads to misreading by DNA polymerase = single base deletion a at GC base pairs

348
Q

How does UV light cause mutations?

A

UV light photons cause adjacent TSH to base pair = dimers with one another

349
Q

Where can IQ be found?

A

Cooked meats and cigarettes

350
Q

What is UVA, B and C important for?

A

UV bActivates Vitamin D in skin
UV b Can cause sun burn and skin cancer
UV a and b together can destroy vitamin a in the skin
UV a b and c can damage collagen fibres and cause skin ageing

351
Q

What mutation repair mechanisms does the body possess?

A

Proof reading
Nucleotide mismatch repair
Excision repair
P53 (guardian angel protein)

352
Q

What is mutation proof reading?

A

DNA polymerase detects mispaired 3’ bases in newly synthesised strand (in DNA replication)

353
Q

What is nucleotide mismatch repair?

A

Enzymes detect bases that don’t base pair in new DNA strand (mismatched)
Enzymes excise and replace the base pairs

354
Q

What is excision repair?

A

Removal of damaged DNA by excision if bases and replacement by DNA polymerase (damaged)

355
Q

What are the two forms of excision repair?

A

Nucleotide excision repair- 30 bases - post UV damage/ carcinogens

Base excision repair - 1 to 5 bases - oxidised, alkylated, delaminates bases and uracil in DNA

356
Q

What is the importance of p53 in repair of mutations?

A

Guardian angel
Monitors repair of damaged DNA
Promotes apoptosis if damage is too severe

357
Q

How can mutations be detected?

A

PCR amplification and southern blotting (e.g. delta FS80 CF- 3 base deletion- phenylalanine lost)
PCR amplification and restriction analysis (e.g. sbs A–>T, restriction site for MstII enzyme destroyed - so one less fragment in mutated gene)
SSCP
Prenatal diagnosis

358
Q

What is the process of SSCP?

A

Mutation scanning
Heterozygous person= mix of normal and mutant sequences
DNA is heated and denatured
It is rapidly cooled - forming a partly double stranded DNA molecule
DNA is electrophoresed on a PA gel
Detected by silver staining

A single nucleotide change in a particular sequence, as seen in a double-stranded DNA, cannot be distinguished by electrophoresis, because the physical properties of the double strands are almost identical for both alleles. After denaturation, single-stranded DNA undergoes a 3-dimensional folding and may assume a unique conformational state based on its DNA sequence. The difference in shape between two single-stranded DNA strands with different sequences can cause them to migrate differently on an electrophoresis gel, even though the number of nucleotides is the same, which is, in fact, an application of SSCP.

359
Q

What are the 3 methods of obtaining foetal DNA in prenatal diagnosis?

A

Amniocentesis (taken from amniotic fluid) 15-20 weeks in ; (0.5-1% rom) ultrasound guidance
Chorion virus biopsy ( trans vascular/-abdominal; foetal virus needs to be separated from maternal tissue) 10-13 weeks in ; ( 2% rom) ultrasound guidance
From mothers blood (still in development stages)

360
Q

What is the most common single base mutation?

A

C to T transition 2/3

361
Q

What does wild type mean?

A

Wild-type – ‘an individual within a population displaying a wild-type trait, which is the trait that is most common in that population’

A mutation causes a mutant phenotype, which is a phenotype that differs from the common or wild type phenotype in the population.

  • A mutation in a gene causes a mutant allele, which is an allele that differs from the common allele in the population (the wild type allele).
  • Mutations that occur in the germline have the possibility of being passed on to offspring – germline mutations.
362
Q

What is the relationship between mutations and cancer?

A

Can be sporadic or heritable
If DNA is damaged to the extent that apoptosis (programmed cell death promoted by p53 protein) doesn’t occur or the damage leads to uncontrolled growth then cancerous cells are produced.

Tumours

  • Tumours are derived from individual abnormal cells.
  • They arise from the lack of normal growth control.
  • Generated by a multistep process.
  • Tumours are more likely to arise from cell types undergoing frequent cell division.
  • All of the cells in a tumour are of the same type.
  • The behaviour of a tumour depends on the cell type.
Oncogenes
Genes involved in control of cell division:
- They’re present in normal cells
- Many different classes
- May stimulate/inhibit growth

Tumour Suppressor Genes
- Genes involved in protecting the cell against one step on the path to cancer.

When the genes are normally present in cells they’re proto-oncogenes. It is after that mutation (or increased expression) that a proto-oncogene becomes oncogene.
Viruses can carry copies of oncogenes, the presence of a virus means that the gene doesn’t function as normal. E.g. HPV.

363
Q

What does sporadic mean?

A

Occurring at random or by chance, and not as a result of a genetically determined, or inherited, trait.

364
Q

What does heritable mean?

A

Capable of being passed from one generation to the next; hereditary

365
Q

What is telocentric?

A

Centromere at top of chromosome

366
Q

What is Acrocentric?

A

Centromere in top portion of chromosome
Chromosome p arms have satellites
Group D and G chromosomes

367
Q

What is submetacentric?

A

Centromere in top portion of chromosome

368
Q

What is meta centric?

A

Centromere in middle of chromosome

369
Q

Describe how genetic info in a cell is organised into chromosomes?

A

Chromosomes are made up of chromatin.
Chromatin is made up of:
- DNA
- Non-histone proteins
- RNA
- Histones (H1. H2A, H2B, H3, H4)
Of the histones H2A, H2B, H3 and H4 interact directly with the DNA. H1 varies between species and H3 and H4 are highly conserved. The histones are responsible for the ‘beads on a string’ structure of chromatin forming nucleosomes.
DNA in the cell is organised into tightly folded chains around histone proteins to form Chromosomes.
In a human cell, there are 23 pairs of chromosomes. These replicate during the S phase of the cell cycle.

370
Q

Describe numerical abnormalities of chromosomes

A 
Numerical – a number of chromosomes other than 46
o Polyploidy (e.g. triploidy, tetraploidy)
o Aneuploidy (an abnormal number that is not a multiple of the haploid number)
371
Q

What is polyploidy?

A

A number of chromosomes which is a multiple of the haploid number and greater than the diploid number of chromosomes

372
Q

What is the main cause of polyploidy?

A

Poly spermy

373
Q

What is poly spermy?

A

Fertilisation of an egg by more than one sper

374
Q

What is aneuploidy?

A

A number of chromosomes which is not a multiple of the haploid number of chromosomes

375
Q

What is monosomy?

A

Type of aneuploidy

Monosomy is a loss of one chromosome i.e. one ‘chromosome pair’ exists as a single chromosome.

376
Q

What is trisomy?

A

Type of aneuploidy

Trisomy is a gain of one chromosome i.e. one ‘chromosome pair’ exists as a triplet

377
Q

Describe structural abnormalities of chromosomes

A

Structural – physical changes to one or more of the chromosomes
Balanced, when the change does not cause any missing or extra genetic info.
Unbalanced, when the changes cause missing or extra genetic info.

378
Q

Describe structural chromosomes abnormalities that exist within one chromosome

A

o Deletion – loss of genetic information
o Duplication – some genetic material is doubled
o Inversion – no loss of genetic material, but a rearrangement of genetic materia
o Ring chromosome – loss of telomeres or ends of both arms and formation of a ring
o Isochromosome – 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.

379
Q

Describe structural chromosomes abnormalities that exist with two chromosomes

A

Inversion – No loss of genetic material, but a rearrangement of genetic material to a non-homologous chromosome
Reciprocal translocation – no loss of genetic material, but an exchange of genetic material between two non-homologous chromosomes
Robertsonian translocation – rearrangement of genetic material between two chromosomes; the q-arms (long) of two acrocentric chromosomes combine to form one ‘super-chromosome’ with the loss of both p-arms.

380
Q

How do you write chromosome nomenclature?

A

The karyotype formula starts with the total number of chromosomes in the cell, followed by a comma, then the X chromosomes, then the Y chromosomes.
E.g. Normal Female = 46,XX and a Normal Male is 46,XY
The plus (+) or minus (-) sign and then a number indicate an extra/missing entire chromosome.
A chromosome number then a p/q and then a +/- indicates an extra/missing piece
E.g. 5p- means ‘missing a segment of the p-arm on chromosome 5

381
Q

How many X chromosomes are active in a cell at any one time?

A

Only one

Other is inactivated and condensed down to form a BARR body seen at periphery of cell nucleus

382
Q

Down’s syndrome?

A

Trisomy 21
More common in boys

3 causes: 
Non disjunction at meiosis : 47, XY, +21
RT 14 & 21 : 46, XY, -14, +t(14q, 21q)
RT 21 & 21 : 46, XY, -21, +t(21q, 21q) 
[N.B DS may arise from a carrier of superchromosomes due to RT]

Symptoms:
Mild to moderate intellectual problems
Congenital heart disease
Haematological malignancies are more common: acute lymphoblastic leukaemia (all)
Hypothyroidism
GI: lack of colon nerves = constipation
Infertility: males infertile, females have reduced fertility
Eye disorders: strabismus, refractive errors, cataracts
Hearing disorders due to infections and malformations

383
Q

Edwards syndrome?

A

Trisomy 18: 47, XX, +18
More common in females (80%)

Modal lifespan: 5-15 days
Mostly diagnosed prenatally

384
Q

Pataus syndrome?

A

Trisomy 13: 47, XY, +13
More than 80% die before they turn 1

Symptoms:
Congenital abnormalities
Polydactyly

385
Q

Turner syndrome?

A

Monosomy X: 45, X
Females

Symptoms:
Short stature
Broad chest
Low hair line
Low set ears
Webbed neck
Cardiovascular symptoms renal problems 
INFERTILITY 
No mental retardation issues
386
Q

Triple X syndrome?

A

Trisomy X : 47, XXX
Females
2/3 X chromosomes are inactive- BARR bodies
Mostly undiscovered

Symptoms:
Tall stature
Microcephaly
Delayed motor skills, speech and learning disabilities
Auditory processing defects
Scoliosis (abnormal curvature of the spine to the sides)
MOSTLY NORMAL FERTILITY

387
Q

Klinefelters syndrome?

A

Trisomy X : 47, XXY
Males
After onset of puberty

Symptoms:
Smaller testes = reduced testosterone production
Glynocomastia (increased breast tissue)
Language, learning and reading impairment (treatment with hormones and surgery)
Reduced facial and pubic hair
INFERTILITY

388
Q

XYY syndrome?

A

Trisomy Y: 47, XYY
Males
Phenotype relatively normal

Symptoms: 
Increase growth rate from early childhood (avg 7cm taller)
Normal testosterone levels
NORMAL FERTILITY
Slightly lower IQ levels
389
Q

Philadelphia chromosome? chronic myelogenous leukaemia (cml)?

A

Specific hormone abnormality associated with chronic myolegenous leukaemia cml
Reciprocal translocation between chromosome 9 & 22
Translocation brings BCR gene on 22 close to ABL1 gene

Results in a protein that makes fusion protein oncogenic

390
Q

Which structural abnormalities are balanced and which are unbalanced?

A

Balanced:
Duplication

Unbalanced:
Interstitial and terminal deletions
Ring chromosome
Isochromosome

Balanced and unbalanced:
Inversion
Reciprocal translocation
Robertsonian translocation

391
Q

Why is it not okay to have Turner syndrome (only one X) when males can survive perfectly fine with one X (XY)?

A

X AND Y chromosomes have short regions in common at the tips of their long or short arms - PSEUDO AUTOSOMAL REGIONS (par1 and par2)
These regions allow the two different chromosomes to recognise one another
Turned syndrome patients will be monosomic for genes in the PARs- has a large effect on phenotype

392
Q

What do the satellite tips of the short arms of chromosomes 14&15 (D) and 21&22 (G) consist of?

A

Duplicated copies of rRNA genes

393
Q

How can deletions be identified?

A

Could be large enough to be seen by standard light microscopy or karyotyping
More cryptic deletions may require FISH

394
Q

What are the main causes of aneuploidy?

A

Non disjunction at meiosis

Anaphase lag

395
Q

What is non disjunction at meiosis?

A

The failure of homologous chromosomes to separate properly during Meiosis 1
OR
The failure of sister chromatids to separate properly during Meiosis 2
Part of chromosome left behind is not destroyed- goes to other cell

396
Q

What is anaphase lag?

A

When a homologous chromosome or sister chromatid is left behind in meiosis which results in such chromosomes being excluded from daughter cell
Due to a defect in spindle function
Part of chromosome left behind is destroyed

397
Q

What are the three causes of Down’s syndrome?

A

Non disjunction at meiosis
Robertsonian translocation 14&21
Robertsonian translocation 21&21

398
Q

In non disjunction at meiosis causing downs - what are the4 possible gametes (with one normal parent)?

A

25% Trisomy 21
25% Monosomy 21
50% normal

399
Q

In RT14&21 causing downs - what are the possible gametes (with one normal parent)?

A

Normal with 14&21 superchromosome (45)
Normal (46)
Monosomy 14 (45)
Trisomy 14 with 14&21 superchromosome (46)
Monosomy 21(45)
Trisomy 21 with 14&21 superchromosome (46)

400
Q

In RT21&21 causing downs - what are the possible gametes (with one normal parent)?

A

Trisomy 21 with 21&21 superchromosome

Monosomy 21

401
Q

What is spermatogenesis?

A

Production of a male gamete- sperm

Cell growth,
Meiosis 1 (homologous chromosomes split into 2 cells)
Meiosis 2 (sister chromatids split into 4 cells) 
Cell differentiation (into sperm- tails etc)
402
Q

What is oogenesis?

A

Production of female gametes

Cell growth
Meiosis 1 (homologous chromosomes split= 1 cell and 1 polar body) 
Meiosis 2 (sister chromatids of cell from meiosis 1 split= 1 ovum and 1 polar body)

In females, primary oocytes enter meiosis 1 before birth and remain arrested there until ovulation - so the later you give birth (maternal age effect) the longer the time available for possible damage to occur to primary oocyte in meiosis 1

403
Q

What is the difference between mosaicism and tetragametic chimerism?

A

Mosaicism- two distinct cell lines (with different DNA) derived from one fertilised egg- caused by non disjunction in one of the early mitotic divisions of the zygote
Tetragametic chimerism- two distinct cell lines (with different DNA) derived from two fertilised eggs which fuse during early embryogenesis (fusion of 2 non identical twins)

404
Q

What is the difference between non disjunction and anaphase lag?

A

Non disjunction- Chromosomes divide unequally over daughter cells
Anaohase lag- Chromosomes left at metaphase plate during anaphase as a result of defective attachment of spindle fibres to the centromere- chromosomes are subsequently lost by degradation in the cytoplasm

405
Q

What is the effect of anaphase lag in mitosis?

A 
Loss of a chromosome
One cell (in which mutation has occurred) and all subsequent daughter cells will be mutant
406
Q

What is the effect of anaphase lag in meiosis?

A

Loss of chromosome
Mutant gamete formed
Consequences for potential offspring

407
Q

What is the importance of a kinase cascade?

A

Amplification of signals by kinase cascades allows amplification of the initial signal by several orders of magnitude within a few milliseconds

408
Q

How are glycogen synthesis and breakdown reciprocally regulated?

A

Epinephrine activated adenylate cyclase
Adenylate cyclase activates ATP to cyclic amp
Cyclic amp activates protein kinase A

(Breakdown) Protein kinase A activates phosphorylase kinase
Phosphorylase kinase activates glycogen phosphorylase

(Synthesis) protein kinase A activates glycogen synthase

409
Q

What are some indications for routine chromosomal investigations?

A
  • Prenatal screening (with maternal age effect ESP-DOWNS)
  • Birth defects (mental retardation/ developmental delay- DOWNS, KLINEFELTERS)
  • Abnormal sexual development (KLINEFELTERS)
  • Infertility (KLINEFELTERS, TURNERS, DOWNS)
  • Recurrent fetal loss (EDWARDS AND PATAUS)
  • Leukaemia (ass with DOWNS cml?)
410
Q

Describe in what aspects transcription and translation in a human cell can be considered ‘similar processes’.

A

 In both processes a template (a code) is read to create a macromolecule built up of basic subunits (a polynucleotide or a polypeptide).
 Both processes consist of three stages: initiation, elongation and termination (of which especially the first and the last stage are highly regulated).
 Both processes require energy and are driven by enzymatic activity.
 In both processes the template has ‘surplus code’ that can be used for regulation; in DNA
promoter sequences, terminator sequences and introns; in mRNA the 5’UTR and 3’UTR.
 The products of both processes undergo further modification before they are ‘fully active’; posttranscriptional modification of RNA (splicing) and posttranslational modification of proteins

411
Q

Describe the key differences between transcription and translation in a human cell.

A

Transcription
an RNA molecule is made takes place in the nucleus a code is copied
(1 unit=1 base to 1 unit= 1 base)

Translation
a protein is made
takes places in the cytoplasm a code is translated
(3 units=triplet -> 1 unit=1 amino acid)

412
Q

What is a polysome?

A

mRNA template covered with very many actively translating ribosomes

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