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Biomed > cell biology S2 Y1 > Flashcards

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)

50
Q

What links pre and post-synaptic membranes?

A

Cadherin (anchored by anchor proteins), neurolignin and neurexin

51
Q

What is the extracellular matrix (ECM)?

A

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

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
53
Q

What does the ECM consist of?

A

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

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)

55
Q

ECM in animals vs plants?

A

Animals = protein (collagen/elastin)
Plants = polysaccharide

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

57
Q

Structure of collagen molecules?

A

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

58
Q

Why are collagen fibres made outside of cells?

A

Too big

59
Q

What prevents collagen fibril assembly?

A

Pro-collagen termini

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

What cross-links stabilise collagen?

A

Lysine-hydroxylysine

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)

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
64
Q

Structure of elastin?

A

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

65
Q

Polysaccharides in matrix in animals vs plants?

A

Animals = glycosaminoglycans (GAGs)
Plants = pectin

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
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
68
Q

Main cell types in ECM?

A

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

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)

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
71
Q

What is adipose tissue?

A

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

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)

73
Q

What is dermis of skin/organ joint capsules?

A

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

74
Q

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

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

What do osteocytes do in bones?

A

Create calcium salts to produce strong structure matrix

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)

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

78
Q

Highly conserved parts of all eukaryotic cells?

A

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

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

80
Q

What is in between the primary wall and plasma membrane?

A

Middle lamella and secondary wall

81
Q

What is rigid cell wall for?

A

Shape and protection from fungi

82
Q

What do plants not have?

A

Intermediate filaments

83
Q

What is the double membrane of the nucleus part of?

A

The ER

84
Q

What is the nucleolus?

A

Site of nuclear ribosome synthesis

85
Q

What are nuclear-pore complexes?

A

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

86
Q

Why do chloroplasts have galactolipids in their cell membranes?

A

So that phosphates can be used for other essential cellular processes

87
Q

What mediates secretory pathways?

A

COP-II coated vesicles

88
Q

What is there more transport of in plants than animals?

A

Sterols to plasma membrane and glycoproteins

89
Q

Why do plant membranes constantly survey their environment?

A

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

90
Q

What mediates the endocytotic pathways?

A

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

91
Q

Where are proteins processed in plants?

A

The systema

92
Q

How are organelles held in place in plant cells?

A

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

93
Q

What do oil bodies do?

A

Spread and house triglycerides as a budding off of the ER

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

95
Q

What are microbodies?

A

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

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
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

98
Q

Examples of plastids?

A

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

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
100
Q

Why does primary cell wall contain cellulose synthases?

A

Produce macromolecular structures

101
Q

Why does primary cell wall contain soluble proteins?

A

Destruction of bacteria

102
Q

Why does primary cell wall contain cellulose microfibrils?

A

Long and provide core strength in protective matrix

103
Q

What is the hemicellulose in primary cell wall?

A

Mesh-like polysaccharide proteins

104
Q

What is the peltin in primary cell wall?

A

Gel-like inter-conecting polysaccharide galacturonans

105
Q

Main role of primary cell wall?

A

Provides support and contains plasma membrane proteins

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

107
Q

Difference in cell wall between plants and fungi?

A

Plants = cellulose-pectin based
Fungi = chitin-based

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

109
Q

What are plasmodesmata for?

A

Osmotic control, communication and contribute to biomechanical sensing and signalling

110
Q

What do plants have for organism flexibility?

A

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

111
Q

What are plasmodesmata?

A

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

112
Q

2 phases of plant life cycle?

A

Vegetative and reproductive

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
114
Q

3 parts of dermal tissue?

A

Cuticle, trichomes (protect) and guard cells

115
Q

3 types of ground/cortex tissues?

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

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

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

Collenchyma:
- What are they?
- Cell walls?

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

Biomed (8 decks)

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