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MGD Flashcards

0
Q

What is the function of the nucleus?

A

RNA synthesis
RNA processing and ribosome assembly
DNA synthesis and repair

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1
Q

What organelles function is for exporting proteins and detoxification reactions?

A

Golgi

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2
Q

What organelles function is to synthesise ATP?

A

Mitochondria

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3
Q

Such organelle synthesises lipids and steroids? Name it’s other functions?

A

Smooth endoplasmic reticulum.
Synthesis cell membrane (phospholipids)
O linked glycosylation
Detoxification reactions

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4
Q

Function of the RER

A

Protein synthesis
Translation
N linked glycosylation

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5
Q

What organelles are there in procaryotic cells?

A

Only ribosomes.
Also have free DNA /RNA, in 1 circular strand
Murein cell wall
Flagella

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6
Q

Name the bond type that holds monomeric units such as amino acids together.

A

Covalent bonds

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7
Q

What types of bonds are macromolecules and complexes held together by?

A 
Non covalent interactions
Hydrogen bonds (electronegative e.g. O N to a H)
Hydrophobic interactions, form bilayers and micelles
Ionic interactions, attraction and repulsion 
Van der waals. Instantaneous induced dipole bonds
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8
Q

Explain the benefits of hydrophobic molecules as storage molecules?

A

Can pass through the lipid bilayer
Doesn’t need to be stored in water
Insoluble in most solutions

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9
Q

A molecule with hydrophobic and hydrophilic properties is said to be

A

Amphipathic

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10
Q

What does pH measure?

A

The concentration of H ions in a solution

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11
Q

The stronger the tendency of an acid to dissociate, the lower the _____ value

A

pKa

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12
Q

What happens when the pH of the solution is higher than the pKa?

A

The deprotonated form dominates
HA + water/soln H+ & A-

Therefore the A- dominates

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13
Q

Low pKa value indicated that the compound is acidic and will easily give up its proton to a base, true or false?

A

True

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14
Q

PKa is calculated when 50% of the compound has dissociated. Use a titration. HA=A-
Calculate the pH for this value,

A

G

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15
Q

How does a buffer work?

A

Reaction is in dynamic equilibrium so that as you add more of OH- for example, it combines with the H+ ion and produces more of the compound HA. This means that is is a bit of an increase in pH but very slight.

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16
Q

Define a buffering region

A

The regions when’re solution can compensate for changes in oH within 1pH of the pKa

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17
Q

Draw an amino acid

A 
Nh3+
Coo-
R
H
China carbon centre
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18
Q

What is the bond that joins amino acids and between which groups does it bond?

A

Peptide bond between the carboxyl group of one amino acid and the amino group of the second.

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19
Q

How do we classify amino acids and give an example of each?

A

According to their side groups, how they interact with water and their polarity, benzene rings
Non polar, aliphatic e.g. Glycerine, alanine, valine
Polar, uncharged side groups e,g, serine, cysteine, glutamine
Aromatic side groups, tyrosine, phenylalanine
Positivly charged side groups, lysine
Negatively charged side groups, glutamate, aspartate

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20
Q

When does the isoelectroc point occur?

A

The pH at which the protein has no overall net charge,
If pH < pI. Then the protein is PROTONATED, accepted H ions like acid
pH > pI then protein is DEPROTONATED

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21
Q

Describe features of an acidic protein

A

NEGATIVELY CHARGED AMINO ACIDS (want to accept H ions)

LOW PI <7

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22
Q

Describe basic proteins

A

Contain many POSITIVE AMINO ACIDs (want to donate protons)

pI>7

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23
Q

Give the typical structures of a secondary protein and their features

A

Alpha helix, 3.6 AA/turn. Right handed. 0.54nm pitch. R groups face out. Bond between c=o and nh3 4 AA away
B pleated sheet. Parallel/anti parallel ( r groups face same direction/opposite)

Hydrogen bonds

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24
Q

How it tertiary structure helpful?

A

Folds the secondary structure so that amino acids far apart in the primary sequence can interact

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25
Q

What it the domain of a protein?

A

Areas in larger proteins (200+) that have distinct structures and serve ow reticular roles. E.g. Ligand binding

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26
Q

Lost bonds involved in tertiary structure

A 
Hydrogen
Van der walls
Ionic
Covalent (disulphide)
Hydrophobic
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27
Q

Describe homomeric and heteromeric proteins

A

Homomeric proteins are quaternary proteins (with more than one polypeptide chain) where the chains are identical
Heteromeric proteins are made of different polypeptide chains
Same bonds involved as in tertiary

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28
Q

Where do disulphide bonds form?

A

Between Cys residues.

Broken by reducing agents

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29
Q

How does pH affect protein structure. And list other things that cal denature proteins

A

Alters the ionisation States of amino acids.
More H ions in solution then it protonates the molecule and alters it. And visa versa
Heat. Vibrations
Detergents. Hydrophobic interactions altered.

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30
Q

What is an amyloidose

A

Cluster of inactive, denatured proteins caused by misfiring proteins. They can clump together and cause disease

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31
Q

What is an amyloid fibre?

A

Misfolded, insoluble form of a normally soluble protein. It is highly ordered and has lots of B sheet.

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32
Q

Proteins can be categorised by either fibrous or globular. Give examples and features for each

A

Globular. Most enzymes and regulatory proteins
Compact, complex structure, several types of 2 structure B barrels and B loops. E.g.haemoglobin

Fibrous. Structure, support, protection
Simple, long strands or sheets of one 2 structure.
E.g. Collagen, alpha chains with h bonds and cross links between.

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33
Q

What is the function of myoglobin and what is its structure and features

A

Binds one molecule of oxygen
Acts as a tempory store of oxygen in the muscle tissue
Not found in the blood stream unless there is a muscle injury
O2 binding causes change in the protein conformation moves the fe into the plane of nitrogens

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34
Q

What is the function of haemoglobin and describe its structure

A

Iron and oxygen binding protein in the red blood cells
Has a tetramer arrangement
2 alpha and 2 beta polypeptide chains
4 molecules of oxygen per haemoglobin molecule

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35
Q

Describe the differences between the two types of deoxygenated haemoglobin

A

T state, tense, low affinity
R state, relaxed, high affinity, easier to bind to
Cooperative binding occurs, becomes easier to bind the next molecule

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36
Q

How does high biphosphoglycerate levels affect binding of oxygen by Hb?

A

High bpg lowers affinity

Shifts curve to right and promotes oxygen release

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37
Q

How does co2 and hydrogen ions affect affinity of Hb for oxygen t

A 
Lower affinity, curve shifts to right
Doesn't pick up as much oxygen in lungs 
Bind to the Hb 
H ions caused by acidic (low pH)
Therfore co2 and h ions produced by metabolically active tissues
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38
Q

How does carbon monoxide act as a poison

A

Binds to haemoglobin 250x more readily
Increases the affinity of Hb for oxygen in unaffected units however this just makes it work as it picks up the oxygen but doesn’t release it

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39
Q

How does sickle cell anaemia occur?

A

Mutation of glutamate to valine in the B chains of haemoglobin
From acidic negative charge
To neutral hydrophobic
Forms a sticky hydrophobic pocket

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40
Q

How does thalassaemia occur?

A

Inbalance between alpha and beta glob in chains

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41
Q

Describe the features of haemoglobin in foetus

A

HbF
Higher binding ability than HbA therfore oxygen transferred to foetus from mother
2 alpha 2 gamma sub chains
No beta Hb

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42
Q

What is the transition state in catalysed reaction

A

High energy intermediate state that occurs during the reaction

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43
Q

What is the Km in the michaelis menten model.

And what does the model represent

A

The substrate concentration that gives half the maximum velocity
Low Km shows that there is a high affinity for the substrate

The rate of enzyme catalysed reaction in relation to concentration of substrate

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44
Q

Describe Vmax

A 
Max velocity (mol/min) 
Max rate when all enzyme active sites are saturated
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45
Q

How do you calculate the rate of reaction using the menten equation

A

Vo(initial relate of reaction)= Vmax [S] / Km +[S]

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46
Q

How can you inhibit enzyme activity irreversibly

A

Drug binds covalently to the enzyme molecule and prevents function

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47
Q

How can an enzyme be inhibited reversible

A

Competitive or non competitive
Competitive: bind at active site, affects km not Vmax, overcome by increasing substrate
Non competitive: binds at another site, affects Vmax not km, not overcome by increasing substrate
Describe IN GRAPH

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48
Q

Describe the main ways of regulating enzymes (3)

A

Substrate and product concentration
Changes in enzyme conformation (allosteric, covalent, proteolytic activation)
Changes in the amount of enzyme, (regulation by synthesis and degradation)

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49
Q

What is an isle stem

A

Different form of the same enzyme
Involved in regulating enzymes
Has different kinetic properties

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50
Q

Describe how allosteric regulation works and give an example

A

Very common
Allosteric activators (enzymes) increase promotion of the r state.
Inhibitors increases t state and shifts curve to left

They do this by an effector binding to a positive or negative alternative active site (allosteric) on the enzyme and change the enzymes shape, therefore Turning off the catalytic activity if negative.
E,g, phosphofructokinase in glycolysis activated by AMP and inhibited by atp, h+ and citrate

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51
Q

Describe covalent modification in controlling enzymes conformation

A

Phosphorylation of the enzyme
Catalysed by kinase
Reversible
Can cause an enzyme cascade

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52
Q

Describe proteolytic activation

A

Inactive protein precursors called zymogens are used to transport the enzyme around the blood to be activated when needed and not before
Activated by removal of part of the chain,e.g. Trypsinogen to trypsin

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53
Q

How can you change the amount of enzyme?

A

Regulation of enzyme syntheisis, transcription and translation
Or of its degradation: targeted for destruction by small proteins, libiquition
Feedback inhibitor
Feed forward activation: increase initial substrate and first step of pathway. And regulation of opposing pathway

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54
Q

Describe the clotting cascade and key regulating steps

A

Clotting factors present as inactive zymogens in the blood
They are activated through a cascade of reactions
The intrinsic pathway is activated when gla binds to the exposed endothelium. Activating factor XII.
This catalyses the next proteolytic activation.
Eventually X to Xa using XII, Ca ions and VII
This catalyses conversion of prothrombin to thrombin which catalyses conversion of fibrinogen to fibrin and uses activated XIII to form a fibrin clot.
Extrinsic pathway activated by tissue damage and activated VII which catalyses Xa production.

Reaction stopped by removal of activated protein, proteolytic digestion and binding of inhibitor molecules

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55
Q

What are nucleotides joined by

A

Phosphodiester bonds, covalent

From the phosphate to the base

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56
Q

Do RNA and DNA strands go from 3 to 5 or 5-3

And which end is the 5’ end and which is the 3’ end?

A

5’-3’
5’ end is the phosphate end
3’ OH on the pentose end. Wrong way :P

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57
Q

What is the difference between ribose and 2 deoxyribose?

A

Ribose has an extra oh group instead of a H on the 2 carbon

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58
Q

List the purine bases, what features do they both have

What do they base pair to in dna and RNA

A

Adenine,
Guanine

Have double rings

Dna: A-T G-C
RNA: A- U g-c

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59
Q

List the pyridamines

A

Cytosine
Uracil (RNA only)
Thymine (DNA only)

G_=C triple bond
A=T or A=U double bond

60
Q

What structure does RNA form

A

Stem loops

Single chain

61
Q

Are solenoid loops visible under light microscopy during cell division when the genes are expressed?

A

Yes
They are made of beads on a string (histones wrapped with DNA nucleosomes) that are all packed into solenoid loops
The loops make up the chromatid.

62
Q

What is a gene

A

Carry the code for protein

Section of the DNS strand in the chain

63
Q

Describe the overall process of somatic cell division

A

G1: cell contents apart from chromosomes replicated
S: chromosomes replicated
G2: chromosome copies checked for errors and repairs made
Mitosis: 2 identical Daughter cells produced,
Cytokinesis

64
Q

Describe what happens during s phase of cell division

A

DNA is replicates.
DNA helix ask unravels the strands, exposing the base pairs, a template strand (initiation)
Nucleotides bind to the exposed bases using DNA polymerase as a catalyst. (Elongation)
Added to the 3’ end
Leading strand synthesised continuously where the lagging strand is made discontinuously (Okazaki fragments) that are joined by ligade
Termination: when two elongations meet (another section unwound) and they join to continue the new strand

65
Q

Describe the main events of mitosis

A

Produces 2 identical daughter cells
Prophase: nuclear envelope breaks down
Pro & Metaphase: spindles attach to chromosomes. They migrate to the centre
Anaphase: spindles move apart and separate the chromosomes
Telophase: nuclear envelope reforms, and two daughter cells produces

66
Q

Describe what is produced in meiosis

A

4 non identical haploid daughter cells produced through 2 divisions
Produces 4 1n sperm
Or 1 egg and 3 polar bodies

67
Q

Describe meiosis and when genetic diversity occurs

A

Prophase
Metaphase, crossing over occurs here, the 2 smaller chromosomes cross over together and the larger ones cross together
Anaphase and telophase then occur
Produces 2 diploid daughter cells with mixed chromosomes
Divide again, :(prophase-telophase)
Overall 4 haploid daughter cells, non identical produces

68
Q

Describe how co-inheritance of certain traits occurs. (Linkage)

A

Genes undergo independent assortment during meiosis

Genes that are close together in the same chromosome often have links and can co segregate
On crossing over they can become separated, the recombination frequency of the links is dependent on the distance between the genes

69
Q

Describe what an allele is

A

The alternative form of the gene. Each individual has 2 alleles for every gene
Can be same or differnt (homo/heterozygous)

70
Q

Describe the process of making proteins from DNA

A

Occurs in the nucleus, synthesis of mRNA from DNA template

Then mRNA translated in the cytoplasm to proteins

71
Q

Describe the process of transcription

A

Initiation: Promoter binds to the template strand, (TATA) which initiates transcription
Elongation: transcription occurs from 5 to 3. Read codon by codon continuously using DNA polymerase to Produce a complementary mRNA. The same happens on the other strand
Termination: sequence dependent termination

72
Q

What modifications are made to mRNA strand?

A

Capping, add a 5’ cap
Polyadentation AAAAAat 3 end
Splicing can then occur to remove the introns, forming mature mRNA
Which acts as the translation template
Then exits (exons) then nucleus to the ribosomes

73
Q

Describe the process of translation

A

Cytoplasm
mRNA to protein
Initiation: start codon AUG, methionine, activates amino acid
Elongation: N to c chain growth, amino acids add nucleotides to the growing polypeptide chain (peptide bonds)
Binds to A site first then moves to P site
Termination: stop codon, prevents further binding

74
Q

What is a polysome

A

Cluster of ribosomes bound to an mRNA molecule

75
Q

List three main types of RNA and how many types of each are there

A

rRNA: few kinds but many copies of each
mRNA: 100000s kinds but few copies of each and only made when needed
tRNA: 100 kinds many copies

76
Q

What is the surplus code section used for regulation in DNA and mRNA?

A

Promoter , terminator sequence and introns in DNA
In mRNA: there’s a 5’ And a 3’ UTR (un translated region outside the open reading frame) in between the ORF and the capping or polyadentation

77
Q

Describe constitutive secretory pathways

A

Protein section that is continuous and not regulated

Proteins packaged into vesicles and released by exocytosis e.g. Collagen

78
Q

Describe regulated secretion of proteins

A

Proteins released in response to signals e.g, hormones

Packaged into vesicles and released after stimuli - insulin

79
Q

Describe how proteins are transported from the ribosome to the nucleus

A

Signal: Nuclear Localising signal (NLS) attaches to protein
Recognised by importin (mediates transport)
Travels through nuclear pore (energy needed)
To nuclei
Signal removed and transported back out using: Ran GTP

80
Q

Describe how proteins are transported from the ribosome to the mitochondria

A

AMPHIPATHIC signal attached on N terminus of the protein
Transported through the double membrane by the channel proteins:
TOM
TIM, using energy
Once in the matrix the signal is then cleaved off

81
Q

Describe how proteins are transported from the ribosome to the lysosomes for exiting the cell

A 
Mannose 6 phosphate signal added post translation
Man 6 P receptor on the trans golgi
Transported in vesicle to the lysine
Signal removes in lysosome
E requires
82
Q

Describe how proteins are transported from the ribosome to the ER

A

Signal sequence on the N terminus
Transported through membrane using a signal recognition protein
Then cleaved off using peptidases signal
E needed

83
Q

Describe how proteins are retained in the ER

A

KDEL signal on the c terminus
Receptor on the cis golgi
Vesicle then delivered
Binds and releases contents by endocytosis
No energy required binding and release dependant on oH

84
Q

Describe the process of protein modification

A

Disulphide bonds: ER
Glycosylation: n (ER) and o linked (golgi)
Trimming and proteolytic processing (golgi)

85
Q

Describe in more detail protein modification in the ER

A

Signal protein cleaved off
N linked glycosylation: add oligosaccharides to the amide group of asparagine
Disulphide bonds then form (cys residues) to increase stability of protein
Then transpired to the golgi,

86
Q

Describe in more detail protein modification in the golgi

A

O linked glycosylation: carbohydrate sugar added using glycosil transferase to the OH group of serine or threonine
Trim n linked proteins
Prolific processing: shorten protein, remove prosegments e.g. Insulin

87
Q

Describe the role of proteolytic processing in the formation of insulin

A

Synthesised as preproinsulin (inactive single polypeptide chain)
Signal sequence removed and 3 disulphide bonds are formed (proinsulin)
Modified again by proteases cut into 3 peptides
Mature insulin: a and b chains held by disulphide bonds and c peptide released

88
Q

Describe how proteolytic processing affects formation of collagen

A 
Lumen of RER 
Signal peptide cleaved off
(Hydroxyl action of proline and lysine residues )
N linked glycosylation 
(Add galactose (carbohydrate))
Disulphide bonds
Form the triple helix structue of 3 alpha chains
O linked glycosylation adding glucose
89
Q

Describe the main features of collagen

A

Triple helix, 3 alpha chains
High tensile strength
Glycine in every 3rd position
H bonds between chains

90
Q

How is restriction analysis used

A

Uses restriction endonucleases to cut the phosphodiester bond of DNA as specific restriction sites.
Cuts the DNa into short fragments of up to 8 base pairs, pallandromic
Gets fragments needed for DNA cloning or further Amplication and analysis
Rejoin fragments using DNA ligase

91
Q

Describe DNA gel electrophoresis

A 
Analysis of small DNA fragments
Buffer solution, 
Power supply
Smaller fragments of DNA travel further toward the anode (positive terminal) 
Uv light used to visualise
92
Q

Explain how PCR is used to analyse DNA

A

Amplifies the DNA sample, millions of copies from template
Use: diagnosis of inherited diseases, tumour? Single base mutations/small deletions/ inversions e,g, cystic fibrosis . infection

93
Q

Describe how PCR occurs

A

1) DNA heated to 95- strands of template denature and separate, h bonds break
2) cooled to 55, primers anneal to target DNA
3) heat to 72- DNA synthesis (hybridisation) occurs using thermo stable DNA polymerase
Repeat 30x ish, exponential growth

94
Q

Are basic proteins positively or negatively charged, and do they go to the anode or cathode in an electric field ee.g. Protein gel electrophoresis

A

Basic proteins - positively charged so to cathode
Acidic proteins - negatively charged to to anode

But depends on size and shape

95
Q

Describe how SDS PAGE (type of gel electrophoresis) is used to separate proteins?

A 
By weight
Denatures the proteins to amino acids
The SDS molecule binds to every 2 AA and masks charge of protein as it is very negative 
Therfore just weight measured 
Use comassie blue dye
96
Q

How is isoelectroc focusing used to study proteins

A

Proteins separated by charge
Applies to pH gradient gel
Proteins migrate until they reach the pH=pI so no overall net charge
More basic, higher pH at top of tube,

97
Q

How is 2D gel electrophoresis used (2d PAGE)

A

Combines SDS page and IEF
Used for complex mixtures
Same pI and different weight or same weight (Mr) and different pI
Diagnosis of disease States in different tissues

98
Q

Why are enzyme assays used

A

To analyse proteins
Act as markers. Measure the activity of the enzyme is it higher or lower than it should be for normal function? Diagnostic tool
Used when optimal conditions, high substrate, suitable pH, temp, ionic strength etc
Metabolic diseases in tissues

99
Q

Give some examples of enzymes assays

A

Aspartate transaminase (ast) marker for liver damage
Amylase and lipase measure for pancritis
Creatine kinase - MI
Lactate dehydrogenase - MI

100
Q

Explain how you can analyse proteins using antibodies. Immuno assays

A

Antibodies can be generated that are highly specific to a protein, can identify a specific protein in a solution
Western blotting: SDS page separates proteins then they are placed on a membrane and specific proteins are visualised by the antibodies binding to it
Enzyme linked immuno absorbent assays (Elisa): the concentration of arotein in a mixture is detected by the binding of the corresponding antibody.

101
Q

Explain how elisa works (analysing proteins using antibodies)

A

The antibody of the protein is immobilised on a solid support - well
The solution nein assayed is washed over
The protein that your looking for is binded to by the anibody and others washed away
A second antibody is added and binds to the antibody/antigen complex and the enzyme used to bind them is measured.
Used to measure insulin and cortisol concentrations

102
Q

Describe the mechanism, specificity, clinical usage and resistance of an antibiotic that inhibits bacterial cell wall synthesis. B lactam antibiotics

A

Penicillin (type of amoxicillin)
Inhibits cell wall (peptidoglycan) synthesis, by binding to the active site of proteins used in facilitating syntheis. so cause death by lysis of bacteria. Only affects actively growing cells.
Only affects bacteria, (not mammal cells, no pep cell wall) affects gut flora though
Clinical use: gram positive bacterial infections, (thicker pep cell wall so greater effect) some gram negative (but they are normally more effective at producing B lactamases), helicobacteria
Resistance: unnecessary usage, e.g. viruses. Not finishing antibiotics, agriculture use
Produce B lactamases that Hydrolyse b lactam ring in the amoxicillin.
Transformation: uptake of resistant gene by a different bacteria and incorporation into the host DNA

103
Q

How do antifolates prevent cell growth? Give an example

A

Methotrexate
Prevents thymidine being produces, preventing folic acid production (which is a cofactor in biosynthesis of RNA and DNA) Therefore anti cancer drug.
Competitive inhibitor to dihydrofolate reductase preventing catalysing dihydrofolate to tetrahydrofolate (folic acid)
Mtx binds more easily.
Less purines produced and less nucleotides produced. Can’t replicate

104
Q

How does resistance to methotrexate occur? Is it specific to mammals? Clinical use?

A

(Impaired transport into cell)
Decreased retention in cell
Increased DHFR action - gene amplification
Mtx specific to mammalian DHFR, as different enzymes for same function
Different antifolate for non mammalian DHFR
Chemotherapy, in high doses
Autoimmune diseases, e.g. Arthritis and chrons disease
Reduces inflammation
Abortions in first 7 weeks

105
Q

Describe the mechanisms of antibiotics that inhibit bacterial transcription? Give an example

A

Rifampicin

Inhibits RNA polymerase therfore no mRNA produced so no translation

106
Q

How do bacteria become resistant to rifampicin

A

Due to misense mutation in structure of the B subunit of RNA polymerase,
changes the binding site of rifamycin on RNA polymerase reducing the affinity for the drug therfore drug less likely to alter it

E.g. TB,MRSA, rickets streptococcus, pneumonia bacteria

Bacteria become resistant through vertical gene transmission (parents to foetus)
Horizontal gene transmission (transformation, transduction, conjugation)
Random mutation
Specific to bacterial RNA polymerase

107
Q

Describe the mechanisms of antibiotics that inhibit bacterial protein syntheis.

A

Bind around the A site on the 30 s ribosome in microbes
Block attwchments of uncharged tRNA to the A site and stops translation
Occurs in gram negative bacteria
E.g. Tetracycline

108
Q

Describe how bacteria develop resistance to tetracycline

A

Horizontal gene transfer between bacteria (not asexual/sexual reproduction)
Leads to 3 genes being produced which produces 3 proteins that do this:
- an efflux pump: pumps tetracycline out of the cell
- ribosomal protection protein - dislodges tetracycline
- tetracycline modification

Specific to 30 s ribosome (therfore not affect our ribosomes)
Treats bacterial infections: urinary tract infections, acne, gonorrhoea, chlamydia, eye infections, pneumonia,

109
Q

Describe southern blotting and when it would be used

A

Studying DNA at gene level
DNA gel electrophoresis
Attached to membrane by blotting and specific fragment visualised using a labelled probe (radioactively/fluorescent) to find the complementary base sequence on the membrane
Occurs using Hybridisation where 2 single stranded complementary DNA sequences reform H bonds between complementary bases.

110
Q

Describe en northern hybridisation/blotting used?

A

Analysing RNA at gene level
Uses an RNA probe when hybridising to analyse
reverse transcriptase PCR: RT PCR
Variant where RNA is used as a template instead of DNA.
Before PCR undertaken, complementary DNA copy is made (cDNA) using reverse transcriptase

111
Q

How can PCR, restriction analysis and DNA hybridisation be used in allele specific tests?

A

Allele specific tests test for known disease causing mutations
In PCR: 2 primers can be used, that are allele specific, and found in one allele or the other
Not all mutations can be detected by PCR, e.g. Partial gene inversions e,g, haemophilia a: need to investigate the gross organisation of the gene. Southern blotting. (Huntingtons, fragile x syndrome)

112
Q

Discuss the range of molecular techniques used to analyse dna at chromosome level

A

Cells arrested in late prophase or early metaphase: condensed but not spread in a line
Then analysed by karyotyping
Or using FISH
Helps detect mutations (incl single base)- common mutations knows and can be tested for specifically using allele specific probes e.g, sickle cell a to t mutation

113
Q

Describe karyotyping and when it is used

A

Analysing chromosome abnormalities
Black and white pic of full set of stained metaphase chromosomes
Visibly look for deformities

114
Q

Describe FISH and when it is used

A

Detect chromosome abnormalities
Fluorescent In Situ hybridisation
Investigates specific DNA sequences on chromosomes inside the cell
Probes for specic gene, or several, label DNA with dye
Denature and hybridise
Reform to double strand
Highlights chromosome
Each chromosome coloured different (paintings)
Investigates: genes (deletions/duplications) chromosome structure, chromosome number and behaviour (anaphase lag)

115
Q

What is array coh used for?

A

Array comparative genomic hybridisation
Screen for sub microscopic chromosomal deletions and duplications and copy number changes when location can’t be deduced from patients phenotype.
Array of DNA probes covering entire genome applied to surface of a solid matrix
Compare it against a reference population which has a different coloured probe attached
Then mix together and can overlay them to compare.
If patients signal show more of the control then shows deletion of chromosome region from where the probe was derived

116
Q

Describe point mutations

A

Point mutation (single base substitution)
Can be:
Transition: purine to purine or pyridamine to pyridamine
Or trans version : pure to pyridamine

117
Q

Describe the effect of mutations on the ORF

A

Insertions and deletions can disrupt the ORF
However if there is a gain or loss of 3bp (codon) maintains reading frame, so there is still a change in the AA code but not the ORF
If in non multiples of 3 there is a frame shift mutation, leading to changes in the code sequence leading to premature termination codons
Can cause a
silent mutation: doesn’t alter as sequence but can disrupt RNA splicing
Misense mutation: can be tolerated in non critical region, i AA substituted by another e.g. GGC to TGC
Nonsense: AA codon to stop codon
Frameshift: ORF of mRNA altered

118
Q

What is a tandem duplication?

A

Insertions or deletions of millions of nucleotides into the sequence of nucleic acid

119
Q

What is the effect of mutations outside the ORF, in the non coding region?

A

Can cause mutations in promotors and start and stop codons

Can change intron splice sites and binding sites of the chain

120
Q

Describe how spontaneous mutations may occur

A

Sequence changed during dna replication, 2 ways:
Tautomeric shift: proton briefly changes position altering bases
Slippage during replication, the new strand forms a small loop leading to the replication becoming out of sync, add another nucleotide on the end to make up the chain.therfore extra

121
Q

Describe how induced mutations may occur

A

Chemicals can alter dna bases, e,g, remove purine rings
Disrupt dna base stacking, disrupts lacking and can lead to single base deletions
Exposure to radiation: e.g, uv damages collagen and destroys vitamin a in skin
Uv photons cause thymine bases to pair with each other.

122
Q

Describe how mistakes in dna synthesis are repaired?

What problems can mistakes lead to?

A

Error rate is high, but most mistakes corrected by DNA polymerase
Missed errors (1%) detected by:
Mismatch repair: post replication enzymes detect and replace thie wrong nucleotide
Excision repair: base excision or nucleotide excision repair. Use different enzymes. Damage Caused by external DNA damage e.g, ROS or uv
Double stranded break repair (DSB) both strands are broken and lead to chromosome rearrangements
Failure - cancers and disease

123
Q

Explain the relationship between DNA damage and cancer

A

Tumours form from cells with 6 new capabilities
To divide independently and indefintely, ignore anti growth signals, avoid apoptosis, invade tissues and establish secondary tumours
Increase probability of successive mutations
Cancer cells have chromosomal and micro satellite instability
BRCA1 and BRCA2 used in detecting dna damage.(common mutations in cancers so can screen for)

124
Q

What are oncogenes?

A

Genes that can transform cells to cancerous cells
Caused by human and animal retroviruses
E,g. HPV 16&18
Human proto-oncogenes can be made after AA substitutions
These can be inherited

125
Q

How is PCR important in diagnosis of genetic disease?

A

Used to amplify small section of DNA for analysis
Identify the mutated region of the gene
Then DNA sequencing
Then PCR of normal and mutated sequences
Form partly double stranded forms

126
Q

Describe main methods of testing for mutations in disease

A

Sequencing for common mutations, using multiplex PCR based test e,g, common cystic fibrosis mutations
More common techniques:
SSCP mutation screening, if a person is heterozygous for mutation then use PCR to get a mix of normal and mutated DNA
Then gel electrophoresis using silver stain. Compare exon locations to see which is the disease causing mutation, there may be other mutations that are not causing the CFTR mutation, so compare with other CF patients

Multiplex ligation dependant probe amplification (MLPA)
Exon counts, spot deletions and duplication . E.g, DMD, osteogenesis imperfecta
Involves hybridisation and PCR
Ethics: if you test child you know results of inherited diseases for you and parents, e.g. Dominant late onset disorders such as Parkinson’s

127
Q

How do we obtain samples for SSCP screening

A

Parental or sibling blood saliva DNA
Prenatal: amniotic fluid cells via amniocentesis 15-20 weeks, using needle
Or chorion villus biopsy 10-13 weeks, sample through uterus
Risk of miscarriage increases slightly
Or foetal DNA from mothers blood, isolate and analyse. But not fully developed yet

128
Q

How are genes expressed?

And how is this substance activated and deactivated

A

Expressed from active chromatin
Chromatin can be activated and deactivated by epigenic modification
Reversible reaction, methylated to inactive
Inactive demethylated to active

No changes to DNA and histones

129
Q

Describe the chromosomal basis of sex determination

A

Sex is determined by the male
During meiosis if the sex cells then have half th number of chromosomes, therfore half of the sperm have an X and half have a Y chromosome
X chromosome inactivation occurs in females as two X chromosomes. The inactivated form remains but not used.

130
Q

How does turners syndrome occur?

A

Chromosomal abnormality of 45, X
One of the X chromosomes is absent or abnormall
Causes short stature, neck webbing, broad chest, sterility, visual impairments,
Susceptible to hypothyroidism, diabetes, autoimmune diseases

131
Q

Describe numerical chromosome abnormalities

A

Aneuploidy: not 46 chromosomes
Loss or gain of a whole chromosome
Caused by malfunction in meiosis divisions.
Leads to gametes having a missing chromosome and an extra one
E.g. Trisomy 21: downs
Polyploidy: gain whole haploid set of chromosomes, caused by polyspermy, e.g. Kleinfelters syndrome 47 XXX 47 XXY

132
Q

Describe structural chromosomal abnormalities

A

Balanced or unbalanced changes
Balanced: exchange/rearrangement of genetic material with not loss or gain of genes
Unbalanced: missing or extra genes caused. Rearrangements within a chromosome

133
Q

List the types of structural chromosome abnormalities and whether they are balanced or unbalanced

A

Deletion: due to uneven pairing or recombination in meiosis, use FISH, unbalanced
Duplication: some genetic material doubled, unbalanced
Inversion: no loss but rearrangement. Can be balanced or unbalanced
Ring chromosome: loss of telomeres or ends of both arms caused ring
Isochromosome: creation of two non identical chromosomes either 2 short arms (p petite) or 2 long arms(q) unbalanced

134
Q

List the types of structural chromosome rearrangements within 2 chromosomes. Balanced or unbalanced

A

Insertions: balanced, no missing or extra genes
Reciprocal translocation: no loss, just exchange between 2 non homologous chromosomes. Carriers produce balanced and unbalanced gametes.
Unbalanced- abnormal phenotype
Robertsonian translocation: rearrangement. Q arm of one chromosome joins to q arm of another- super chromosome. E.g, 14&21 chromosome count is 45 in balanced carriers. There is a risk of aneuploidy. Homologous carriers can be phenotypic ally normal but will produce downs offspring

135
Q

Describe the main features of a Karyotype and how they are ordered

A

Ordered from large to small A-G
Stained by trypsin and romanowski dye
Dark g positive bands are AT rich and gene poor
Light g negative bands are GC rich and gene rich (lighter and richer gold coins)
Use ISCN nome culture to describe

136
Q

Why would patients be referred to have a Karyotype done?

A

For an accurate diagnosis
Frequent miscarriages
Developmental delay
Acquired reasons: leukaemia, solid tumours
Prenatal diagnosis
Asses future risks, future reproduction risks

137
Q

How is FISH important in detecting chromosomal abnormalities

A

Looks for specific mutations,
Probes hybridise to specific genes, loci, centromeres, telomeres, while chromosomes
Add probe, denature to single strand, hybridise, re anneal with probe, wash, visualise

138
Q

How is prenatal aneuploidy screening used

A 
Genetic test for abnormal chromosomes
Interphase analysis with fish
Scans for common aneuploidies within 48 hrs
Can then do a full scan later if needed
Abortion?
139
Q

How is microarray methodology used in chromosome abnormalities.
Explain advantages and disadvantages

A

Detects unbalanced chromosomes- detects copy number changes, unbalanced. And covers the entire genome, in detail,
Doesn’t detect rearrangements for example,mosaicism sometimes missed
Uses DNA not chromosomes- compare normal control to sample
Red green comparison. Where colour is different there is a deletion or duplication
Expensive

140
Q

Describe mosaicism

A

Presence of 2 or more cell lines in an individual
Caused by Mitotic non disjunction
If occurs in fist post zygote can division- looks like meiotic event and no mosaicism
Next divisions - 3 cell lines

141
Q

describe uni parental disomy (UPD)

A

Presence of homologous chromosomes from 1 parent
Isodisomy: 2 identical chromosomes from 1 parent (meiosis 1 error) can lead to duplication of lethal recessive diseases, potentially dangerous
Heterodisomy: 2 homologous chromosomes from 1 parent (meiosis 2 error)
Or part of s chromosome from one parent
And none from the other

Mostly phenotype is not affected,
However rare recessive disorders can occur
Can lead to angelman syndrome, or prader willi syndrome for example (chromosome 15 affected)

142
Q

Explain how UPD occurs, with reference to trisomy and monosomy rescue

A

Meiotic error occurs in first division leading to one cell having 2 chromosome gametes
Causes a trisomy when cells combine
Then mitosis occurs (protozygotic mitosis) causing a cell with 2 chromosomes and a cell with normal 1 chromosome
1 in 3 chance

143
Q

When doing pedigrees, what are the two things to look for? As a way to tell what inherited

A

Is there anyone whose phenotype is different to that of both their parents? Yes? Then x linked or autosomal recessive

Are there any girls with the recessive PHENOtype, who have a father or a son with the dominant phenotype?
Yes then can’t be x linked because the father needs to have the recessive X chromosome for the daughter to have inherited XX recessive.
No then could be x linked, if father and son have the diseased phenotype.

144
Q

Describe a strong acid.
Ka
Donor or acceptor or protons

A

Strong acids High ka value
As strong acids donate protons easily
Have negative amino acid groups

145
Q

When pH < PI then….

A

Protein is protonated (accepts H and neutralises the acidic amino acid groups)

146
Q

Describe the process of gram staining

A

Cells stained with crystal violet dye
Iodine aded
Acetone or methanol added as a decolourizer
Crystal violet is trapped in thicker gram positive cell wall
It degrades the thinner peptidoglycan cell wall therfore decolourizing it
Red dye (safranin) is added. And stained the gram negative red

147
Q

Describe how antipyreitics work in temperature reduction

A

Inhibit cyclo oxygenase enzyme

Reduce levels of prostaglandins (PGE) within hypothalamus

148
Q

How is dna replicated

A

Initiation: recognition and binding to an origin or replication. Dna helicase unzips the dna double helix. Primers laid down by RNA primase
Elongation: replication from 5-3 in leading and lagging strands.,lagging is discontinuous and leads to Okazaki fragments
Termination: RNA primers removed,discontinuous fragments joined by DNA ligase
Semi conservative replication: in the 2 daughter helices one is parental DNA and other is from newly synthesised DNA

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