cell biology S2 Y1 Flashcards

1
Q

What determines migration in gel electrophoresis?

A

Size and shape of nucleic acid

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2
Q

What is standard agarose gel electrophoresis for?

A

Medium-sized nucleic acids

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3
Q
  • What is polyacrylamide gel electrophoresis for?
  • Stains?
A
  • Smaller nucleic acids due to its higher resolution
  • Ethidium bromide and SYBR gold
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4
Q

How is polyacrylamide made?

A

Adding acrylamide and methylene bisacrylamide with persulfate TEMED

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5
Q
  • Role of formamide?
  • Why does DNA not need it?
A
  • Linearises single stranded nucleic acid chain as without it they take specific structural conformations
  • Double-stranded so it is linear and has no particular shape
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6
Q

Southern blotting:
- What does it enable?
- 7 steps?

A
  • Determination of presence of particular DNA species
    1. Gel electrophoresis carried out
      1. Gel is put in salt solution
      2. Overlayed with nylon membrane which soaks up salt that is absorbed by gel
      3. This transfers the DNA
      4. Put into a bag with radioactive probes
      5. They hybridise to complimentary sequence
      6. X-rayed to create autoradiogram
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7
Q

Northern blotting:
- What is it?
- 5 steps?

A
  • Same as Southern blotting but analyses RNA
    1. RNA extracted from tissues
      1. mRNA fraction is isolated using OligodT dynabeads
      2. Purified mRNA is then formamide-denatured
      3. Run on urea-polyacrylamide gel for blotting
      4. Radioactively labelled probe applied that is complimentary to mRNA
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8
Q

What is pulse field electrophoresis?

A

Type of electrophoresis that increases the length that can be identified by alternating the direction of current application so DNA changes direction during migration - longer DNA will take longer to turn so more separation observed in longer (500kB-1MB)

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9
Q

What is electrophoretic mobility shift assay (EMSA)?

A

Differentiates between free DNA and DNA bound to protein by synthesising DNA/protein of interest and subjecting it to assay with native PAGE conditions (free DNA moves fastest)

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10
Q

Single strand conformational polymorphism (SSCP) electrophoresis:
- Advantage?
- 4 steps?
- Way of proving mutation?

A
  • Quick
    1. Have mutant and non-mutant DNA
      1. Both heated to denature
      2. Rapidly put on ice
      3. Single stranded structures form without going back to double-stranded
  • Mutant DNA will run at different rate to non-mutant if it is actually mutated
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11
Q

What is transfection?

A

Process of introducing naked nucleic acids into eukaryotic cells

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12
Q

Transient transfection:
- What does it use?
- How does transfection occur?
- How does it occur naturally in jellyfish?

A
  • Liposomes that have nucleic acids put in the centre, then nucleic acid and lipofection reagent combined to form complexes and transfection occurs (then assayed)
  • Liposome fuses with the membrane of cell and empties contents
  • Promoter is upstream which is an expression vector, and there is a multiple cloning site downstream that allows cloning of gene of interest and fusion with protein of interest
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13
Q

Stable transfection:
- How does it work?
- What performs it?
- Disadvantages?
- 2 enzymes involved?

A
  • DNA integrated into host genome and expressed with genome so it is not degraded by nucleases (unlike in transient transfection)
  • Retrovirus-mediated infection
  • Laborious and long
    1. Reverse transcriptase (makes DNA copy of RNA genome)
      1. Integrase (puts DNA into genome)
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14
Q

Stable transfection:
- Role of 2 vectors involved?

A
  • One males virus particles
  • One makes RNA copies –> has a tag so particle thinks it is viral genome –> causes release –> growth medium applied and virus released into target cell (DNA enters) (TRANSDUCTION)
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15
Q

What is a transgenic model of disease?

A

Relevant gene is inserted to an organism’s genome to show genetic disease is caused by this

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16
Q

6 steps of making transgenic model of disease?

A
  1. Start off with gene targeting vector with a selection marker
  2. Transfected into cell
  3. Vector undergoes homologous recombination with host chromosome
  4. Gene replacement occurs
  5. Produces modified target gene with selection marker
  6. Successful mutant cells are inserted into blastocyst
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17
Q

2 roles of the selection marker?

A
  1. Disrupts target gene
  2. Enables selection of cells that have undergone mutagenic recombination
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18
Q

How are unsuccessful cells left out of insertion into blastocyst?

A

Selected against using a resistance marker

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19
Q

How is a particular gene deleted in a specific tissue/organ?

A

Cre-LoxP-based methodology (controlled knockout)

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20
Q

How does Cre-LoxP work?

A

LoxP sites are either side of two inverted repeats with a spacer separating them, Cre-recombinase induces recombination and deletes the sequence to create a floxed allele

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21
Q

Haemophilia A:
- What causes it?
- Symptom?
- Treatment?

A
  • Mutations in gene that encodes factor VIII
  • Excessive bleeding as factor VIII involved in blood clotting
  • Factor VIII replacement therapy
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22
Q

7 steps of obtaining factor VIII?

A
  1. Stable transfection of CHO/BHK cells with engineered viral expression vectors for human factor VIII
  2. Factor VIII production in hamster cells
  3. Stringent purification of factor VIII
  4. Inactivation of any potentially contaminating viral particles
  5. Nano-filtration to remove any viral particles and/or other potentially contaminating pathogens
  6. Quality control
  7. Market
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23
Q

Main disadvantage of hamster-made factor VIII and how was this tackled?

A

Patients became immune through immunogenic reaction that produced an inhibitor as hamsters had different post-translational modifications to the factor than humans - tackled by using human HEK293-F cells

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24
Q

Why does single stranded DNA move at different rates in SSCP?

A

Nucleotide substitutions cause different ssDNA shapes that change base pairings and interaction

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25
Q

Why does SSCP use non-denaturing conditions?

A

Denaturing conditions break molecular bonds so shape changes (but SSCP needs shapes to be informative)

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26
Q

Why does a labelled digonucleotide allow most definitive assessment of the presence of a DNA species in a Southern blot?

A

It is complimentary

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27
Q

How is a mutation introduced into a mammalian cell line?

A

Retrovirus-mediated infection

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28
Q

What are epithelia?

A

Avascular tissues composed of cells and organised into sheets or tubules (attached to an underlying ECM basement membrane)

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29
Q

2 types of epithelia? Further divisions?

A

Simple or stratified (columnar, cuboidal or squamous)

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30
Q

7 roles of epithelia?

A
  1. Mechanical properties (skin)
  2. Permeability barrier (small intestine)
  3. Absorption (small intestine)
  4. Filtration (epithelium of renal corpuscle)
  5. Secretion (sweat glands)
  6. Diffusion of gases and fluids (lung alveoli)
  7. Sensory (retina)
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31
Q

Why are epithelium polarised?

A

Can transport molecules in directional manner

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32
Q

How are epithelia specialised?

A

Morphologically (creates very different types) e.g. apical membrane split into microvilli OR basal membrane has basal lamina

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33
Q

How are epithelia held together?

A

By cell junctions

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34
Q

4 types of cell junctions?

A
  1. Anchoring junctions (link cells together or to extracellular matrix)
  2. Occluding junctions (seal gaps between cells)
  3. Channel forming junctions (create passageways to link cytoplasm of adjacent cells)
  4. Signal relaying junctions (allow signals to be communicated cell to cell)
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35
Q

2 ways anchoring junctions link cell to cell?

A
  1. Adherins junction - attached to actin filaments with cadherin, alpha-caterin and beta-caterin
  2. Desmosome - attached to intermediate filaments with cadherin and plakoglobin desmoplakin
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36
Q

2 ways anchoring junctions link cell to basal lamina?

A
  1. Focal adhesions - attached to actin filaments with integrin and focal adhesion kinase
  2. Hemidesmosome - attached to intermediate filaments with integrin, collagen and dystonin
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37
Q

Role of cadherins?

A

Mediate cell-cell attachment and link and are attached to a cytoskeletal filament in each cell

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38
Q
  • What do adherins junctions link cadherins to?
  • What do desmosomes “ “?
A
  • Actin filaments
  • Intermediate filaments
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39
Q

How do epithelial sheets form tubes/vesicles?

A

Adhesion belt (associated with actin) undergoes organised tightening to cause invagination, then epithelial tube pinches off overlying sheet

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40
Q

Role of integrins?

A

Play central role in mediating cell-matrix contacts

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41
Q

What do hemidesmosomes do?

A

Anchor epithelial cells to basal lamina

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42
Q

What can defective desmosomes cause?

A

Pemphigus vulgaris (autoimmune destruction of desmosomal protein)

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43
Q

3 roles of occluding junctions?

A
  1. Seal gaps between apical cells
  2. Fence function (prevents free diffusion) - maintains polarity
  3. Barrier prevents free flow (prevents Crohn’s)
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44
Q

Type of junctions in occluding junctions?

A

Tight with zona occludin scaffold protein (also have a network of strands with homophilic interactions)

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45
Q

How can the fence function of occluding junctions going wrong cause cancer?

A

Cells lose polarity and contract = metastasis

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46
Q

Role of channel forming junctions?

A

Allow ions and small molecules to pass from cell to cell

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47
Q
  • Type of junction in channel forming junctions?
  • What can defects cause?
A
  • Gap made up of connexons which have 6 subunits that form a cylinder with gap in the centre
  • Cataracts, vokwinkel syndrome
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48
Q

Difference in location of tight and gap junctions?

A

Tight - near apical surface
Gap - closer to apical lamina

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49
Q

Role of signal-relaying junctions?

A

Allows communication of signals between cells (synapses)

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50
Q

What links pre and post-synaptic membranes?

A

Cadherin (anchored by anchor proteins), neurolignin and neurexin

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51
Q

What is the extracellular matrix (ECM)?

A

Any substance produced by cells and secreted into extracellular space within tissues

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52
Q

2 roles of the ECM?

A
  1. Structural - physical support and linkage between cells and tissues
  2. Cell motility - forms track for substrate cells to move on and provides cues to guide direction for movement
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53
Q

What does the ECM consist of?

A

Collagen fibres that are cross-linked by accessory proteins in a matrix of proteoglycans

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54
Q

Where is ECM in relation to epithelial tissues?

A

Little in the tissue as it is just layers of cells closely bound to one another, instead it is concentrated under epithelia in basal lamina (creates base for cells to sit on, acts as molecular sieve and substrate for migrating cells)

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55
Q

ECM in animals vs plants?

A

Animals = protein (collagen/elastin)
Plants = polysaccharide

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56
Q

What does ECM make in plants?

A

Cell wall - made up of cellulose fibres cross-linked with hemicellulose in a matrix of highly branched polysaccharides

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57
Q

Structure of collagen molecules?

A

Triple-stranded (cross-linked by proteins) and form collagen fibrils that form collagen fibres

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58
Q

Why are collagen fibres made outside of cells?

A

Too big

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59
Q

What prevents collagen fibril assembly?

A

Pro-collagen termini

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60
Q
  • Cross-links between components of tropocollagen subunit?
  • Between fibrils?
A
  • Covalent
  • Hydroxyproline
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61
Q

What cross-links stabilise collagen?

A

Lysine-hydroxylysine

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62
Q

What are collagen helical regions made up of?

A

GLY -X - Y
(X and Y are any amino acid but usually Pro/HydroxyPro)

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63
Q

9 steps of collagen fibre formation?

A
  1. Pro-alpha chain synthesis
  2. Hydroxylation of selected prolines + lysines
  3. Glycosylation of selected hydroxylysines
  4. Self-assembly of three pro-alpha chains
  5. Procollagen triple-helix formation
  6. Secretion via secretory vesicle
  7. Cleavage of procollagen to form collagen
  8. Self-assembly into collagen fibril
  9. Aggregation of collagen fibrils to form a collagen fibre
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64
Q

Structure of elastin?

A

Large filaments with random coils that are connected by cross-linked lysine and hydroxlysine

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65
Q

Polysaccharides in matrix in animals vs plants?

A

Animals = glycosaminoglycans (GAGs)
Plants = pectin

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66
Q

Glycosaminoglycans:
- Charge?
- What are they linked to?
- Do they take up large of small amounts of space?
- What do they link?

A
  • Negative + very hydrophillic (absorb H2O)
  • Non-fibrous proteins called proteoglycans
  • Large but efficiently
  • Proteins that hold aggrecan to a hyaluronan molecule and are bound to proteoglycans
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67
Q

4 ways connective tissue varies?

A
  1. Proportion of fibres to cells in ECM
  2. Number and proportion of different cell types in ECM
  3. Proportion and arrangement of fibres in ECM
  4. Composition of non-fibrous component of ECM
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68
Q

Main cell types in ECM?

A

Fibroplasts, macrophages, mast cells, osteocytes (bone), chrondrocytes (cartilage), adipocytes (fat), blood cells

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69
Q

What is stretchy skin caused by?

A

Failure of conversion of lysine to hydroxylysine by lysyl hydroxylase OR failure to cleave off propeptide termini (fibrils+fibres do not form)

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70
Q
  • Role of areolar connective tissue?
  • Structure?
A
  • A loose connective tissue that links organs, adds support+strength+elasticity
  • Criss-cross of elastin and collagen
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71
Q

What is adipose tissue?

A

Adipocytes held together by reticular fibres (collagen III) that stores fat

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72
Q

What are tendons and ligaments?

A

Collagen dominant tissue with all collagen in orientation of direction of force from the muscle it is attached to (resist forces)

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73
Q

What is dermis of skin/organ joint capsules?

A

Many orientations of collagen due to unpredictable force direction (resist tension)

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74
Q

Elastic cartilage:
- Made up of?
- What secretes cartilage components?

A
  • Elastin in many orientations against distortion
  • Chrondrocytes in the lacuna
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75
Q

What do osteocytes do in bones?

A

Create calcium salts to produce strong structure matrix

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76
Q

What is scurvy?

A

Lack of vitamin C (an essential cofactor for hydroxylases that make HydroxyPro and Lys - needed for crosslinking of collagen fibres - damages fibrils + synthesis)

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77
Q

What is fibrodysplasia ossificans progressiva?

A

Muscles and connectives tissues ossified to become bone
Caused by mutation in ACVR1/ALK2 encoding activin A receptor activin-like kinase 2

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78
Q

Highly conserved parts of all eukaryotic cells?

A

Peroxisomes, plasma membrane, nucleus, endomembrane system, ribosomes, mitochondria, oil body, cytoskeleton

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79
Q

Why do plant cell membranes have no cholesterol and more sterols?

A

Allows rapid changes in response to changing environment e.g. temperature

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80
Q

What is in between the primary wall and plasma membrane?

A

Middle lamella and secondary wall

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81
Q

What is rigid cell wall for?

A

Shape and protection from fungi

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82
Q

What do plants not have?

A

Intermediate filaments

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83
Q

What is the double membrane of the nucleus part of?

A

The ER

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84
Q

What is the nucleolus?

A

Site of nuclear ribosome synthesis

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85
Q

What are nuclear-pore complexes?

A

High order quaternary protein complex aggregates that act as supramolecular sieves that control export and import

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86
Q

Why do chloroplasts have galactolipids in their cell membranes?

A

So that phosphates can be used for other essential cellular processes

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87
Q

What mediates secretory pathways?

A

COP-II coated vesicles

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88
Q

What is there more transport of in plants than animals?

A

Sterols to plasma membrane and glycoproteins

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89
Q

Why do plant membranes constantly survey their environment?

A

To recycle receptors and have the most relevant ones on their membrane

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90
Q

What mediates the endocytotic pathways?

A

Clathrin and COP-I coated vesicles
(receptors remoulded and recycled by heat-shock proteins)

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91
Q

Where are proteins processed in plants?

A

The systema

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92
Q

How are organelles held in place in plant cells?

A

Tethering by the ER (uses specific accessory proteins -desmotubule, transvacuolar strand)

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93
Q

What do oil bodies do?

A

Spread and house triglycerides as a budding off of the ER

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94
Q

What are ER-tonoplast and ER-chloroplast said to be?

A

Semi-autonomous as they can grow and undergo fission without the cell cycle
BUT THEY CONTAIN DNA

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95
Q

What are microbodies?

A

Semi-autonomous organelles than house molecular components, BUT HAVE NO DNA

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96
Q

3 examples of microbodies and what they are?

A
  1. Oil bodies = 1 layer, buds off of ER
  2. Peroxisomes = house catalase, mops up reactive oxygen from photosynthesis
  3. Glyoxysomes = store fatty acids that are processed into CoA
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97
Q

What are plastids?

A

Semi-autonomous cells that make their own DNA and ribosomes, have a double membrane made of galactolipids and move around the cytosol via actin

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98
Q

Examples of plastids?

A

Etioplast, chromoplast, chloroplast, leucoplast, amyloplast, elaioplast, proteinoplast

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99
Q

What does ___ contain:
- Embryo?
- Seed?
- Seedling?
- Mature leaf?
- Senescent leaf?

A
  • Chloroplasts
  • Oil bodies and proteins for embryo to use for growth and making new plasma membranes
  • Etioplasts and stores glyoxysomes
  • Leaf peroxisomes and mitochondria
  • More oil bodies and etioplasts due to degradation of compounds
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100
Q

Why does primary cell wall contain cellulose synthases?

A

Produce macromolecular structures

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101
Q

Why does primary cell wall contain soluble proteins?

A

Destruction of bacteria

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102
Q

Why does primary cell wall contain cellulose microfibrils?

A

Long and provide core strength in protective matrix

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103
Q

What is the hemicellulose in primary cell wall?

A

Mesh-like polysaccharide proteins

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104
Q

What is the peltin in primary cell wall?

A

Gel-like inter-conecting polysaccharide galacturonans

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105
Q

Main role of primary cell wall?

A

Provides support and contains plasma membrane proteins

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106
Q

What is the difference between the secondary cell wall and primary?

A

Secondary has tighter arrangement of cellulose microfibrils and hemicellulose, also contains lignin which are water-impermeable polyphenolic molecules that add rigidity and support

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107
Q

Difference in cell wall between plants and fungi?

A

Plants = cellulose-pectin based
Fungi = chitin-based

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108
Q

Difference in junctions for adhesion and communication in plants and animals?

A

Plants = plasmodesmata (everything held in
cell wall)
Animals = tight+gap junctions, desmosomes

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109
Q

What are plasmodesmata for?

A

Osmotic control, communication and contribute to biomechanical sensing and signalling

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110
Q

What do plants have for organism flexibility?

A

Partially permeable primary cell walls stuck together with gel-like middle lamella

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111
Q

What are plasmodesmata?

A

Desmotubules that link to the ER with complex, branched architecture to maintain cell wall integrity

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112
Q

2 phases of plant life cycle?

A

Vegetative and reproductive

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113
Q

3 types of permanently vegetative cells/tissues?

A
  1. Ground cells of the cortex
  2. Vascular cells of vascular bundles
  3. Epidermal/dermal cells of the dermal tissue
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114
Q

3 parts of dermal tissue?

A

Cuticle, trichomes (protect) and guard cells

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115
Q

3 types of ground/cortex tissues?

A
  1. Parenchyma
  2. Collenchyma
  3. Sclerenchyma
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116
Q

Parenchyma:
- Level of specialisation?
- Role?
- Cell walls?

A
  • Low
  • Perform key metabolic functions
  • Have thin and flexible primary, no secondary
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117
Q

Collenchyma:
- What are they?
- Cell walls?

A
  • Differentiated parenchyma
  • Thicker and more uneven primary, no secondary
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118
Q

Sclerenchyma:
- What are they?
- Cell walls?
- 2 types?

A
  • Differentiated parenchyma that die once secondary walls are laid down
  • Thick secondary
    1. Sclerids = short, irregular, thick, lignified secondary walls
    2. Fibers = long, slender, arranged in threads
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119
Q

2 types of xylem cells?

A
  1. Tracheids = in all plants, long, thin, tapered ends, promote lateral water movement
  2. Vessel elements = in tall trees, lignified, perforated end walls, no end plates, promotes upward water movement
120
Q

What are sieve tube elements?

A

Tube-like cells that conduct nutrients and have a porous-end sieve, are alive at functional maturity but have no nucleus, ribosomes, vacuole or cytoskeleton

121
Q

What happens when a signal molecule binds to a receptor?

A

Signalling downstream cascade in cell for response and activation of intracellular signalling molecules

122
Q

What do cell surface receptors bind to?

A

Signal molecules that cannot pass through the plasma membrane

123
Q

What do intracellular receptors bind to?

A

Small, hydrophobic signal molecules within the cell as they can pass through the plasma membrane

124
Q

Difference between fast and slow responses?

A

Fast have rapid protein alterations to change function
Slow express new genes and form new proteins (alters protein synthesis)

125
Q

What do phosphate kinases do?

A

Attach phosphate groups

126
Q

What do phosphate phosphatases do?

A

Remove phosphate groups

127
Q

How are GTP-binding proteins activated and inactivated?

A

Activated upon GTP binding
Inactivated upon GTPase activity as it hydrolyses GTP to GDP

128
Q

How do GTP-binding proteins inactivate themselves?

A

Via intrinsic GTPase activity

129
Q

What do trimeric GTP-binding (G) proteins do?

A

Relay signals from G-protein-coupled receptors

130
Q

What do monomeric GTPases do?

A

Assist other cell surface receptors

131
Q

Crucial role of G-protein coupled receptors (GPCRs)?

A

Transmitting signals from external environment to inside of cells

132
Q

What physiological processes are GPCRs involved in?

A

Sensory perception, neurotransmission, hormone regulation, immune response

133
Q

Types of signal molecules that bind to GPCRs?

A

Proteins, small peptides, derivatives of amino/fatty acids

134
Q

Typical structure of GPCRs?

A

Have 7 hydrophobic transmembrane domains (single polypeptide chain that threads back and forth across lipid bilayer)

135
Q

When are trimeric G proteins activated?

A

When an extracellular signal molecule binds a GPCR and it conformationally changes

136
Q

General structure of trimeric G proteins?

A

Have alpha, beta and gamma protein subunits (2 of which are tethered to the plasma membrane by short lipid tails)

137
Q

What are trimeric G proteins like in their resting state?

A

Tethered to inner leaflet of plasma membrane, alpha subunit is associated with a GDP molecule

138
Q

GPCRs:
- What does alpha subunit cause?
- What then happens?
- What is the time the subunits are activated for equivalent to?
- What does alpha subunit control?

A
  • Decrease of GDP affinity so it is exchanged for GTP = separation of alpha from beta and gamma subunits
  • Alpha subunit interacts with target protein
  • How long response will be for
  • Time (hydrolyses GTP to GDP so subunits recombine and response ends)
139
Q

2 main roles of trimeric G proteins?

A
  1. Directly regulate ion channels
  2. Activate membrane-bound enzymes which produce small messenger molecules
140
Q

What does the trimeric G protein regulation of ion channels cause?

A

Immediate change in state and behaviour of the cell

141
Q

How is the heartbeat an example of trimeric G proteins regulating ion channels for a response?

A

GPCR activates trimeric G protein and the beta-gamma complex binds to the intracellular face of a K+ channel in the plasma membrane of pacemaker cells, forces ion channel into open conformation and then as it opens the heart is slowed as the plasma membrane’s permeability to K+ increases, so it is harder to electrically activate
Original signal is terminated and the K+ channel closes when the alpha subunit inactivates itself

142
Q

Why are interactions between activated trimeric G proteins and enzymes slower and more complex?

A

As they lead to production of additional intracellular signalling molecules

143
Q

What are small molecules generated by enzymes upon trimeric G protein binding called?

A

Second messengers (amplify and spread the intracellular signal)

144
Q

What does trimeric G protein activation of adenylyl cyclase produce?

A

cAMP from ATP

145
Q

What terminates cAMP signal?

A

Cyclic AMP phosphodiesterase converts cAMP to AMP

146
Q

What do high levels of cAMP activate?

A

Cyclic-AMP-dependent protein kinase (PKA) as it changes conformation of regulatory protein that holds PKA and releases an active form of PKA

147
Q

2 things activated PKA catalyses the phosphorylation of?

A
  1. Cytoplasmic enzymes to change their activity
  2. Transcription factors that can alter gene expression
148
Q

How does adrenaline binding to a GPCR cause glycogen breakdown?

A

Signal causes alpha subunit to bind GTP (activate) and then bind adenylyl cyclase so cAMP is formed – binds to inhibitor proteins on PKA and a phosphorylation cascade activates phosphorylase and glycogen phosphorylase so glycogen breaks down

149
Q

What is an example of trimeric G proteins directly regulating ion channels?

A

Vision (GPCRs called rhodopsin are utilised)

150
Q

How does image processing in the eye work?

A

The rhodopsin GPCR activates transducin trimeric G protein which activates cyclic GMP phosphodiesterase which breaks down cGMP to 5’ GMP to decrease cytosolic [cGMP] that means that less cGMP-gated channels are open in plasma membrane so hyperpolarisation occurs (less Na+ and Ca2+ enters) and this blocks the release of inhibitors that act on cells further along pathway = perceived light can reach brain before being processed to an image to be seen

151
Q

What are receptor-tyrosine kinases?

A

A type of enzyme-coupled receptor with a cytosolic domain with intrinsic kinase activity (phosphorylates amino acids)

152
Q

What activates receptor-tyrosine kinases?

A

Ligand binding to extracellular domain

153
Q

What do receptor-tyrosine kinases mediate?

A

Cell growth, survival and differentiation

154
Q

RTK activation:
- What happens when ligand binds?
- What then happens?
- What does the phosphorylated tyrosine become?

A
  • Pulls domains together through autophosphorylation
  • More phosphate groups added to tyrosine residues on the outside of the kinase domain
  • A docking site for adaptor/signalling proteins
155
Q

3 main types of downstream RTK signalling?

A
  1. Ras/MAPK pathway
  2. PI3K-Akt pathway
  3. Phospholipase C-gamma pathway
156
Q

Ras activation:
- What does Ras need to interact with RTK?
- Role of Grb2?
- What does Ras-GEF act as?
- What is GTP binding?
- What is GTP hydrolysis?

A
  • GEF and Grb2
  • Activates Ras-GEF and interacts with RTK
  • Molecular signalling switch that is membrane bound via lipid side chain
  • Ras-GEF promoted, GDP out and GTP in
  • Ras-GAP promoted, self-inactivating, phosphate out
157
Q

MAP kinase cascade:
- What is it?
- What controls it?
- What is it an example of?
- 2 results to promote cell proliferation?

A
  • A conformational signalling switch
  • Phosphorylation status
  • Amplification
  • Protein phosphorylation or transcription factor phosphorylation
158
Q

MAP kinase cascade:
- Protoncogenes?
- Concogenes?
- When are proteins active?

A
  • Normal Ras and MAPK genes
  • Constitutively activated versions of them, regardless of phosphorylation status
  • When phosphorylated
159
Q

PI3K activation:
- What does activated RTK with PI3K bound use PIP2 for?
- What is PIP3 used as?
- What phosphorylates Akt?
- What does Akt activation mean and why?

A
  • To form PIP3
  • Binding site on membrane for protein kinase 1 and Akt
  • Protein kinases
  • Cell survival as Akt phosphorylates Bad which releases active Bcl2 to inhibit apoptosis AND cell growth occurs via mTOR activation
160
Q

PLC pathway:
-What does activated RTK with PLC-gamma bound use PIP2 for?
- What is the fate of IP3?
- Role of DAG?

A
  • To generate DAG and IP3
  • Released into cytosol and binds and opens Ca2+ channels in ER to increase [Ca2+] in cytosol
  • Recruits and activates protein kinase C and Ca2+ binds to PKC and causes phosphorylation of target proteins
161
Q

What do ligand-gated ion channels need to open?

A

Specific chemicals to bind

162
Q

What are gated ion channels responsible for?

A

Signalling between nerve cells as they convert chemical signals to electrical signals

163
Q

Role of voltage gated ion channels in nerve cell signalling?

A

Generate and carry action potential along axon to the presynaptic terminal

164
Q

Role of ligand gated ion channels in nerve cell signalling?

A

Rapid transmission of signals across synapses in the nervous system via neurotransmitter binding to channel on postsynaptic membrane

165
Q

Where is the acetylcholine receptor?

A

Between motor neuron and skeletal muscle cell

166
Q

Glycine transmitter gated ion channel:
- What does it mediate?
- What does it process?
- When is it open?
- What happens when it is open?

A
  • Fast inhibitory nerve signalling in CNS
  • Processes motor and sensory information to allow movement and vision
  • When glycine is bound
  • Cl- enters –> hyperpolarisation –> harder for threshold to be reached = lower chance of action potential (inhibitory)
167
Q

What is necrosis?

A

Unplanned cell death that is usually in response to damage or infection (can cause further damage to surrounding cells and tissues)

168
Q

How does apoptosis avoid issues associated wirh necrosis?

A

Dismantles cells

169
Q

What occurs in S phase?

A

DNA replication

170
Q

What occurs in G1 and G2 phases?

A

Growth and monitoring of internal+external environments to ensure conditions are suitable for replication

171
Q

3 things a cell does during interphase?

A
  1. Transcribes genes
  2. Synthesises proteins
  3. Grows in mass
172
Q

3 cell cycle checkpoints?

A
  1. In M phase to check chromosomal attachment to the mitotic spindle
  2. End of G1 to do environment checks for replication
  3. End of G2 to check DNA is fully replicated and there is no DNA damage
173
Q

When must DNA damage be fixed before?

A

DNA replication and the M phase

174
Q

What regulates the start transition in late G1 phase?

A

Outside influences

175
Q

What occurs when cells signal that more cells are needed?

A

Cell proliferation

176
Q

Why can cancer result from cell cycle?

A

Malfunction in regulation of cell numbers can result in cancerous growth

177
Q

CDKs:
- What part of cell cycle relies on them?
- Where are they present?
- Does concentration change?
- Role?
- How does activity rise and fall?

A
  • Progression
  • Dividing cells
  • No
  • Phosphorylate proteins in the cell cycle when activated (cyclin bound)
  • In/activated by cyclin binding
178
Q

Does cyclin change concentration through cell cycle and why is this important?

A

Yes as they drive cyclic assembly and activation of cyclin-CDK complexes

179
Q

What do different cyclin-CDK complexes trigger?

A

Different steps in the cell cycle

180
Q

Why does cyclin concentration change?

A

Rises are due to transcription of cyclin genes and formation of cyclin proteins, falls are due to degradation

181
Q

What does cyclin degradation help with?

A

Driving entry into the next cell cycle phase

182
Q

2 pathways for cells when they exit the cell cycle?

A
  1. Undergo apoptosis
  2. Enter G0
183
Q

What are quiescent cells?

A

Cells withdrawn to G0, but can reenter the cell cycle under certain conditions

184
Q

What is terminal differentiation?

A

Permanent withdrawal from the cell cycle (or in G0)

185
Q

What is senescence?

A

Termination of cell division

186
Q

What is immortalisation/transformation?

A

The evading of senescence cancer cells do

187
Q

What is a benign tumour?

A

Excessive growth of cells in original tissue, less dangerous but can become malignant

188
Q

What is a malignant tumour?

A

Cancerous, cells invade surrounding tissue

189
Q

What are metastases?

A

Cancerous cells breaking down boundaries, entering the bloodstream to take over distant sites and form these metastases

190
Q

2 contributors to cancer development?

A
  1. Activation of proto-oncogene
  2. Inactivation of a tumour suppressor gene
191
Q

3 gene mutation pathways that can occur in individual tumours?

A
  1. Signals to induce cell proliferation
  2. Governing of cell division cycle
  3. Regulation of cell survival in response to DNA damage and stress
192
Q

What do the proteins cyclin-CDK complexes phosphorylate play a role in?

A

Initiation of DNA replication, nuclear envelope breakdown, chromosome separation

193
Q

What destroys cyclins?

A

Ubiquination (at a point of no return from one stage to the next)
- APC/C targets the proteins for destruction, inactivates cyclin-CDK complexes and then ubiquitylation occurs for destruction

194
Q

How many times must DNA be replicated in cell division?

A

ONLY ONCE

195
Q

What is the proliferating cell nuclear antigen (PCNA) part of?

A

DNA replication complex - PCNA is a sliding clamp

196
Q

How is it ensured DNA only replicates once in the S phase?

A

Origins of replication only fire once and then the cyclin-CDK complexes are targeted for destruction by APC/C

197
Q

What happens when APC/C is removed?

A

Origins of replication keep firing and the cells re-replicate the DNA but never divide

198
Q

What do homologous chromosomes replicate to form?

A

Sister chromatids

199
Q

What holds sister chromatids together until separation?

A

Cohesin rings

200
Q

What are microtubules responsible for?

A

Polarising cell via forming bipoles (begins in S-phase with duplication of centrioles and centrosomes)

201
Q

What is a centrosome?

A

Centrioles and a mass of proteins called the pericentriolar material

202
Q

What is a centriole?

A

A microtubule array

203
Q

3 steps of centriole duplication in S-phase?

A
  1. Firstly disengaged
  2. Duplicated into pro-centriole which elongates and matures into daughter centriole
  3. Centrosomes separate into 2 mother and daugher groups
204
Q

Prophase:
- Role of condesins?
- How is mitotic spindle formed?

A
  • Loop chromatin into tight bundles
  • Centrosomes move apart
205
Q

Prometaphase:
- What attaches to microtubules?
- How does the nuclear envelope break down?

A
  • Condensed chromosomes
  • Lamins are phosphorylated by cyclin B-CDK1 and the nuclear pore complexes disassemble
206
Q

Metaphase:
- Where do sister chromatids line up?
- What is dynamic instability?
- What orientates mitotic spindle?

A
  • Metaphase plate
  • Microtubules grow slowly and shrink rapidly
  • Microtubule physical force
207
Q

Kinetochores:
- What are they?
- What do sensor proteins do?

A
  • Protein complexes made of structural and signalling proteins that link chromatin and microtubles
  • Monitor attachment of microtubules and sense tension on microtubules
208
Q

Spindle assembly checkpoint:
- What does it do?
- Role of APC/C?
- Role of separase?

A
  • Inhibits APC/C when kinetochores are exposed
  • Degrades securin
  • Cleaves cohesin
209
Q

What occurs in anaphase?

A

Cohesins holding together sister chromatids are degraded

210
Q

Telophase:
- What do microtubules do?
- What begins to form on midline?

A
  • Bundle and push newly formed nuclei apart
  • A contractile ring to cleave the cells
211
Q

What occurs in cytokinesis?

A

Contractile ring cinches and pinches, midbody forms at scission point

212
Q

What forms when cytokinesis fails?

A

Binucleate cells

213
Q

What does oocyte meiosis arrest in until ferilisation?

A

Metaphase II

214
Q

How do homologous chromosomes pair?

A

Via complimentary DNA sequences

215
Q

How do 2 pairs of sister chromatids swap genetic material?

A

Form a four-chromosome bivalent joined by synaptonemal complex (synapsis) that allows swapping

216
Q

4 ways genetic diversification can occur?

A
  1. Independent assortment
  2. Crossing over (segment swapping)
  3. Gene conversion (non-crossing over homologous recombination)
  4. Non-disjunction (chromosome segregation going wrong)
217
Q

Why does meiosis I only separate the homologous chromosomes?

A

Spindle body pulls one side of each chromosome so they stay together due to chiasma (but pairs split)

218
Q

Why does meiosis II separate sister chromatids?

A

Spindle bodies pull either side

219
Q

4 benefits of compartmentalised genetic material in eukaryotes?

A
  1. Protects DNA
  2. Increase surface area for membrane-localised reactions
  3. Increased efficiency of transcription and synthesis
  4. More ways to regulate gene expression
220
Q

What is the inner membrane of the nucleus continuous with?

A

The lumen of the ER

221
Q

How did the nucleus evolve?

A

Nuclear DNA was attached to the membrane and then engulfed to form the nucleus

222
Q

What are nuclear lamins?

A

Meshwork of filaments (related to intermediate filaments) on inner nuclear membrane that associate with DNA

223
Q

How are nuclear lamins connected to the cytoskeleton?

A

By SUN/KASH proteins in inner membrane
By nesprins in outer membrane

224
Q

What is chromatin?

A

DNA and RNA complex organised around nuclear matrix

225
Q

Difference between euchromatin and heterochromatin?

A

Euchromatin is less visible, less dense and around the centre of the nucleus
Heterochromatin is darker, tightly packed and around lamina and nucleoli

226
Q

What does discrete territories of chromosomes in nucleus allow?

A

Co-regulation of genes on different chromosomes (depending on orientation)

227
Q

Why are Lamina-Associated Domains (LADs) rich in heterochromatin?

A

Due to the low level of transcription

228
Q

What are nucleoli?

A

Non-membrane bound organelles in the nucleus

229
Q

5 functions of nucleolus?

A
  1. Transcription of rRNAs
  2. Assembly of ribosomal subunits
  3. Sensing and responding to stress
  4. Cell cycle regulation
  5. Cancer
230
Q

What segments of chromosomes does the nucleolus contain?

A
  1. Encode rRNA genes
  2. Proteins for processing rRNAs
  3. Ribosomal subunit proteins
  4. Small nucleolar RNA oligonucleotides
231
Q

3 other nuclear structures?

A
  1. Cajal bodies
  2. PML bodies
  3. Speckles
232
Q

What are cajal bodies?

A

Proteins that process RNA, organise the genome and have a dense foci of coilin protein

233
Q

What PML bodies?

A

Proteins associated with cajal bodies and are involved in DNA repair, cell proliferation and apoptosis

234
Q

What are speckles?

A

Proteins associated with gene transcription and mRNA processing (splicing)

235
Q

What is exported out of the nucleus?

A

mRNA, rRNA, tRNA and assembled ribosomal units

236
Q

What is imported into the nucleus from the cytoplasm?

A

RNA polymerase, ribosomal proteins, transcription factors, all other structural proteins

237
Q

What are nuclear pore complexes?

A

A ring structure with 8-fold symmetry that allows things in and out and acts as a selective barrier formed by FG-Nups in central channel

238
Q

What is required for the facilitated transport of mRNA into the nucleus?

A

ATP hydrolysis

239
Q

What is required for the facilitated transport of proteins, tRNA and ribosomes into the nucleus?

A

GTP hydrolysis

240
Q

What is required for the facilitated nuclear transport?

A

Nuclear transport receptors called karyopherins

241
Q

How is mRNA exported out of the nucleus?

A

As a mRNA-protein (mRNP) complex that includes processing, capping and splicing proteins as well as export factors

242
Q

Why does mRNA transport require ATP hydrolysis?

A

ATP hydrolysis releases Dbp5 in cytoplasm and triggers mRNP dissociation –> the energy is needed to maintain a transport gradient

243
Q

What gradient is formed for protein import/export?

A

Gradient of Ran protein in GDP and GTP bound form whereby Ran-GDP is in the cytosol and Ran-GTP is in the nucleus (Ran-GAP in cytosol, Ran-GEF in nucleus)

244
Q

How do GTPases act as molecular switches?

A

Bind GTP and hydrolyse a phosphate group to form GDP (off)

245
Q

What is GTP hydrolysis assisted by?

A

GAPs

246
Q

What does GTP loading after hydrolysis?

A

GEFs

247
Q

Role of importins?

A

Facilitate protein nuclear import by binding cargo proteins in cytoplasm and interacting with FG-Nups to release cargo into the nucleus (cargo release is enabled by Ran-GTP binding to importin-beta as complex comes apart)
- Importin and Ran-GTP then goes back out to the cytosol

248
Q

Role of exportins?

A

Facilitate protein nuclear export and they bind to cargo proteins, interact with FG-Nups
- Cargo loading is enabled by Ran-GTP binding to exportin (complex assembly)

249
Q

What are nuclear localisation signals (NLSs)?

A

Protein motif anywhere on protein that binds to importins

250
Q

What are nuclear export signals (NESs)?

A

Sequence with four hydrophobic amino acids that bind to exportins

251
Q

How can NLSs and NESs be modified?

A

By phosphorylation

252
Q

How does nucleocytoplasmic shuttling work?

A

NLS of the NF-kB transcription factor is masked when bound to the lkB inhibitor in the cytoplasm until a signal like cytokine triggers degradation of the inhibitor to expose NLS

253
Q

How does a translocation event give rise to leukaemia?

A

Chromosome 9/22 swap at breakpoint cluster region causes BCR and ABL genes to fuse which creates a constitutively active kinase that becomes an oncogen and in an overproliferated, stem-like state that can resist apoptosis

254
Q

Why do humans have 23 pairs of chromosomes but all other primates have 24?

A

Humans had a telomere-telomere fusion event

255
Q

What shows DNA is not lost during development?

A

Animal cloning as transferred nucleus gives rise to whole organism (gene expression must be regulated and DNA can be reprogrammed following differentiation)

256
Q

5 steps of animal cloning in sheep?

A
  1. Female has egg removed and enucleated
  2. Udder cells removed and placed in culture and grown in G1 stage
  3. Cell transferred into enucleated egg via electrofusion
  4. Cultured into blastocyst
  5. Blastocyte implanted into surrogate mother
257
Q

7 considerations when choosing an animal model?

A
  1. Ease to maintain
  2. Cost
  3. Known genome sequence
  4. Ease of breeding
  5. Experimental advantages
  6. Ethical considerations
  7. Animal licence
258
Q

3 advantages of an animal model when studying genetics?

A
  1. Large number of offspring
  2. Short gestation
  3. Large array of mutants
259
Q

3 advantages of an animal model when studying embryology?

A
  1. Many embryos
  2. External development
  3. Robust but easy to manipulate embryos
260
Q

3 advantages of an animal model when studying genomics?

A
  1. Relevance to human genome
  2. Disease models
  3. Drug testing
261
Q

7 steps of frog life cycle?

A
  1. Gemetogenesis
  2. Fertilisation
  3. Cleavage
  4. Gastrulation
  5. Organogenesis
  6. Larval stages
  7. Maturity
262
Q

How does cleavage form 8 blastomeres?

A

Central cleavage from animal to vegetal pole then right-angled cleavage and then perpendicular cleavage

263
Q

What do fertilised frog eggs have?

A

A grey crescent opposite where sperm entered as cortical cytoplasm rotates 30 degrees

264
Q

4 steps of gastrulation?

A
  1. Firstly blastocoel is present (space in centre)
  2. Then invagination (blastopore) forms at dorsal lip
  3. Then involution and epiboly occur to start archenteron development
  4. Archanteron and blastocoel coexist and a yolk plug forms
265
Q

3 germ layers?

A
  1. Ectoderm (outer)
  2. Mesoderm (middle)
  3. Endoderm (internal)
266
Q

What is neuralation?

A

Notochord signals neural plate to form a groove that invaginates into a neural tube

267
Q

What type of cleavage occurs in mammals, amphibians and insects?

A

Mammals = rotational
Amphibians = radial
Insects = superficial

268
Q
  • What is holoblastic cleavage?
  • 5 types?
A
  • Whole egg divided
  • Radial, spiral, bilateral, rotational and radial
269
Q
  • What is meroblastic cleavage?
  • 3 types?
A
  • Part of egg divided (lots of yolk)
  • Bilateral, discoidal, superficial
270
Q

Where are cells smallest after cleavage in amphibians?

A

At the top

271
Q

Where are there more blastomeres in amphibians?

A

Animal pole

272
Q

How do mammal eggs cleave?

A

Asynchronistically and not at the same time, do not divide at 90 degrees (meridionally and equatorially instead)

273
Q

How does superficial cleavage work in insects?

A

Nuclei divide but not the cell and nuclei move to the edge of the cell in the syncitial blastoderm stage and cellularisation (cell division) occurs

274
Q

5 types of cell movements during gastrulation?

A
  1. Invagination = inpocketing of cells forms
  2. Involution = whole sheet of cells moves together inside embryo to go up and over roof of embryo
  3. Ingression = single cells move away from outer edge of embryo to inside
  4. Delamination = cells move from outside of embryo to inside as a sheet
  5. Epiboly = cells spread and cover entire early embryo
275
Q

How do inductive signals work in patterning?

A

Early embryo has a signalling centre that sends an inductive signal along the embryo to create transcription factor code that creates 3 new body regions in the late embryo

276
Q

How does induction work?

A

Cell secretes an inducing signal, closest cells have high signal, further have low/no signal

277
Q

How do transcription factors work?

A

Repress or activate expression of genes by binding to DNA elements

278
Q

How do induction signals affect transcription factors?

A

Different factors affected by different signals (more signal = greater effect = different cell types)

279
Q

How does blastula formation occur?

A

Through cleavage

280
Q

What germ layer gives rise to the nervous system?

A

Ectoderm

281
Q

What does cleavage immediately follow?

A

Fertilisation

282
Q

What is mesoderm?

A

Germ layer that makes bones and muscle

283
Q

What is a blastopore?

A

Structure that forms first embryo opening

284
Q

What is neurulation?

A

Process that forms notochord and neural tube in Xenopus laevis embryos

285
Q

What is induction?

A

Process where one group of cells/tissues influences development of neighbouring cells/tissues

286
Q

What is forward genetics?

A

Starts with random mutation –> find phenotype –> find gene

287
Q

What is reverse genetics?

A

Start with gene –> remove gene –> look at phenotype

288
Q

Genetic screen:
- What are they?
- 3 steps?

A
  • Forward genetics technique that identifies and studies an interesting phenotype in a mutated population
    1. Generate random mutants
    2. Observe phenotype
    3. Identify mutated genes
289
Q
  1. Generating mutants
    - Who are mutagenised?
    - What then happens?
    - What are the F1 crossed with?
    - Why are the F2 generation then inbred?
A
  • Males
  • Mated with females to form some normal offspring and some mutant +/- (not on same gene) offspring
  • Wild type to generate normal or heterozygous mutants in F2 generation
  • To generate F3 that 1/16 are homozygous (-/-) mutants
290
Q
  1. What phenotypes are observed?
A

Loss-of/gain-of-function

291
Q
  1. Identify the gene:
    - 6 steps?
    - How is RNA location in the embryo identified?
A
    1. Embryos fixed
    2. RNA probe added and labelled with a tag
    3. Antibody labelled with enzyme added
    4. Embryos washed
    5. Substrate reacts with enzyme to form coloured precipitates
    6. Shows where RNA is in embryos
  • RNA probe binds to the RNA and antibody with tag binds to this
292
Q
  • What is genetic engineering?
  • 4 examples?
A
  • A reverse genetics technique that modifies gene expression
  • Transgenesis, targeted knockouts, CRISPR-Cas9, RNAi
293
Q

Transgenesis:
- What is it?
- How does it work?

A
  • Addition of a transgene (gene of interest) to see effect via plasmid DNA containing the gene of interest and a detection marker
  • DNA is transcribed via injection into fertilised oocyte (held in place via holding pipette) into the male pronucleus –> the litter is then screened for transgenic animals
294
Q

7 steps of targeted knockouts (removal of a gene)?

A
  1. Generate a targeting vector (expression of DNA of interest interrupted by adding foreign DNA to coding region)
  2. Obtain embryonic stem cells (must be undifferentiated to form any cell type)
  3. Genetically modify ES cells (targeting vector electroshocked into nuclei and homologous recombination occurs with host chromosome to form modified target gene)
  4. Inject modified ES cells into blastocysts to form recombinant embryo
  5. Implant recombinant embryos into surrogates (uterine horn in mice)
  6. Screen for chimeric pups
  7. Breed the chimeric mice with ES cell donor mice
295
Q

What are chimera?

A

Mixture of normal and targetted cells

296
Q

What are heterozygous knockout offspring?

A

Offspring presenting recessive trait from ES cell donor

297
Q
A