cell biology S2 Y1 Flashcards
1
Q
What determines migration in gel electrophoresis?
A
Size and shape of nucleic acid
2
Q
What is standard agarose gel electrophoresis for?
A
Medium-sized nucleic acids
3
Q
- What is polyacrylamide gel electrophoresis for?
- Stains?
A
- Smaller nucleic acids due to its higher resolution
- Ethidium bromide and SYBR gold
4
Q
How is polyacrylamide made?
A
Adding acrylamide and methylene bisacrylamide with persulfate TEMED
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
6
Q
Southern blotting:
- What does it enable?
- 7 steps?
A
- Determination of presence of particular DNA species
- Gel electrophoresis carried out
- Gel is put in salt solution
- Overlayed with nylon membrane which soaks up salt that is absorbed by gel
- This transfers the DNA
- Put into a bag with radioactive probes
- They hybridise to complimentary sequence
- X-rayed to create autoradiogram
- Gel electrophoresis carried out
7
Q
Northern blotting:
- What is it?
- 5 steps?
A
- Same as Southern blotting but analyses RNA
- RNA extracted from tissues
- mRNA fraction is isolated using OligodT dynabeads
- Purified mRNA is then formamide-denatured
- Run on urea-polyacrylamide gel for blotting
- Radioactively labelled probe applied that is complimentary to mRNA
- RNA extracted from tissues
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)
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)
10
Q
Single strand conformational polymorphism (SSCP) electrophoresis:
- Advantage?
- 4 steps?
- Way of proving mutation?
A
- Quick
- Have mutant and non-mutant DNA
- Both heated to denature
- Rapidly put on ice
- Single stranded structures form without going back to double-stranded
- Have mutant and non-mutant DNA
- Mutant DNA will run at different rate to non-mutant if it is actually mutated
11
Q
What is transfection?
A
Process of introducing naked nucleic acids into eukaryotic cells
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
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
- Reverse transcriptase (makes DNA copy of RNA genome)
- Integrase (puts DNA into genome)
- Reverse transcriptase (makes DNA copy of RNA genome)
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)
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
16
Q
6 steps of making transgenic model of disease?
A
- Start off with gene targeting vector with a selection marker
- Transfected into cell
- Vector undergoes homologous recombination with host chromosome
- Gene replacement occurs
- Produces modified target gene with selection marker
- Successful mutant cells are inserted into blastocyst
17
Q
2 roles of the selection marker?
A
- Disrupts target gene
- Enables selection of cells that have undergone mutagenic recombination
18
Q
How are unsuccessful cells left out of insertion into blastocyst?
A
Selected against using a resistance marker
19
Q
How is a particular gene deleted in a specific tissue/organ?
A
Cre-LoxP-based methodology (controlled knockout)
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
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
22
Q
7 steps of obtaining factor VIII?
A
- Stable transfection of CHO/BHK cells with engineered viral expression vectors for human factor VIII
- Factor VIII production in hamster cells
- Stringent purification of factor VIII
- Inactivation of any potentially contaminating viral particles
- Nano-filtration to remove any viral particles and/or other potentially contaminating pathogens
- Quality control
- Market
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
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
25
Why does SSCP use non-denaturing conditions?
Denaturing conditions break molecular bonds so shape changes (but SSCP needs shapes to be informative)
26
Why does a labelled digonucleotide allow most definitive assessment of the presence of a DNA species in a Southern blot?
It is complimentary
27
How is a mutation introduced into a mammalian cell line?
Retrovirus-mediated infection
28
What are epithelia?
Avascular tissues composed of cells and organised into sheets or tubules (attached to an underlying ECM basement membrane)
29
2 types of epithelia? Further divisions?
Simple or stratified (columnar, cuboidal or squamous)
30
7 roles of epithelia?
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)
31
Why are epithelium polarised?
Can transport molecules in directional manner
32
How are epithelia specialised?
Morphologically (creates very different types) e.g. apical membrane split into microvilli OR basal membrane has basal lamina
33
How are epithelia held together?
By cell junctions
34
4 types of cell junctions?
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)
35
2 ways anchoring junctions link cell to cell?
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
36
2 ways anchoring junctions link cell to basal lamina?
1. Focal adhesions - attached to actin filaments with integrin and focal adhesion kinase
2. Hemidesmosome - attached to intermediate filaments with integrin, collagen and dystonin
37
Role of cadherins?
Mediate cell-cell attachment and link and are attached to a cytoskeletal filament in each cell
38
- What do adherins junctions link cadherins to?
- What do desmosomes " "?
- Actin filaments
- Intermediate filaments
39
How do epithelial sheets form tubes/vesicles?
Adhesion belt (associated with actin) undergoes organised tightening to cause invagination, then epithelial tube pinches off overlying sheet
40
Role of integrins?
Play central role in mediating cell-matrix contacts
41
What do hemidesmosomes do?
Anchor epithelial cells to basal lamina
42
What can defective desmosomes cause?
Pemphigus vulgaris (autoimmune destruction of desmosomal protein)
43
3 roles of occluding junctions?
1. Seal gaps between apical cells
2. Fence function (prevents free diffusion) - maintains polarity
3. Barrier prevents free flow (prevents Crohn's)
44
Type of junctions in occluding junctions?
Tight with zona occludin scaffold protein (also have a network of strands with homophilic interactions)
45
How can the fence function of occluding junctions going wrong cause cancer?
Cells lose polarity and contract = metastasis
46
Role of channel forming junctions?
Allow ions and small molecules to pass from cell to cell
47
- Type of junction in channel forming junctions?
- What can defects cause?
- Gap made up of connexons which have 6 subunits that form a cylinder with gap in the centre
- Cataracts, vokwinkel syndrome
48
Difference in location of tight and gap junctions?
Tight - near apical surface
Gap - closer to apical lamina
49
Role of signal-relaying junctions?
Allows communication of signals between cells (synapses)
50
What links pre and post-synaptic membranes?
Cadherin (anchored by anchor proteins), neurolignin and neurexin
51
What is the extracellular matrix (ECM)?
Any substance produced by cells and secreted into extracellular space within tissues
52
2 roles of the ECM?
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
What does the ECM consist of?
Collagen fibres that are cross-linked by accessory proteins in a matrix of proteoglycans
54
Where is ECM in relation to epithelial tissues?
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
ECM in animals vs plants?
Animals = protein (collagen/elastin)
Plants = polysaccharide
56
What does ECM make in plants?
Cell wall - made up of cellulose fibres cross-linked with hemicellulose in a matrix of highly branched polysaccharides
57
Structure of collagen molecules?
Triple-stranded (cross-linked by proteins) and form collagen fibrils that form collagen fibres
58
Why are collagen fibres made outside of cells?
Too big
59
What prevents collagen fibril assembly?
Pro-collagen termini
60
- Cross-links between components of tropocollagen subunit?
- Between fibrils?
- Covalent
- Hydroxyproline
61
What cross-links stabilise collagen?
Lysine-hydroxylysine
62
What are collagen helical regions made up of?
GLY -X - Y
(X and Y are any amino acid but usually Pro/HydroxyPro)
63
9 steps of collagen fibre formation?
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
Structure of elastin?
Large filaments with random coils that are connected by cross-linked lysine and hydroxlysine
65
Polysaccharides in matrix in animals vs plants?
Animals = glycosaminoglycans (GAGs)
Plants = pectin
66
Glycosaminoglycans:
- Charge?
- What are they linked to?
- Do they take up large of small amounts of space?
- What do they link?
- 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
4 ways connective tissue varies?
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
Main cell types in ECM?
Fibroplasts, macrophages, mast cells, osteocytes (bone), chrondrocytes (cartilage), adipocytes (fat), blood cells
69
What is stretchy skin caused by?
Failure of conversion of lysine to hydroxylysine by lysyl hydroxylase OR failure to cleave off propeptide termini (fibrils+fibres do not form)
70
- Role of areolar connective tissue?
- Structure?
- A loose connective tissue that links organs, adds support+strength+elasticity
- Criss-cross of elastin and collagen
71
What is adipose tissue?
Adipocytes held together by reticular fibres (collagen III) that stores fat
72
What are tendons and ligaments?
Collagen dominant tissue with all collagen in orientation of direction of force from the muscle it is attached to (resist forces)
73
What is dermis of skin/organ joint capsules?
Many orientations of collagen due to unpredictable force direction (resist tension)
74
Elastic cartilage:
- Made up of?
- What secretes cartilage components?
- Elastin in many orientations against distortion
- Chrondrocytes in the lacuna
75
What do osteocytes do in bones?
Create calcium salts to produce strong structure matrix
76
What is scurvy?
Lack of vitamin C (an essential cofactor for hydroxylases that make HydroxyPro and Lys - needed for crosslinking of collagen fibres - damages fibrils + synthesis)
77
What is fibrodysplasia ossificans progressiva?
Muscles and connectives tissues ossified to become bone
Caused by mutation in ACVR1/ALK2 encoding activin A receptor activin-like kinase 2
78
Highly conserved parts of all eukaryotic cells?
Peroxisomes, plasma membrane, nucleus, endomembrane system, ribosomes, mitochondria, oil body, cytoskeleton
79
Why do plant cell membranes have no cholesterol and more sterols?
Allows rapid changes in response to changing environment e.g. temperature
80
What is in between the primary wall and plasma membrane?
Middle lamella and secondary wall
81
What is rigid cell wall for?
Shape and protection from fungi
82
What do plants not have?
Intermediate filaments
83
What is the double membrane of the nucleus part of?
The ER
84
What is the nucleolus?
Site of nuclear ribosome synthesis
85
What are nuclear-pore complexes?
High order quaternary protein complex aggregates that act as supramolecular sieves that control export and import
86
Why do chloroplasts have galactolipids in their cell membranes?
So that phosphates can be used for other essential cellular processes
87
What mediates secretory pathways?
COP-II coated vesicles
88
What is there more transport of in plants than animals?
Sterols to plasma membrane and glycoproteins
89
Why do plant membranes constantly survey their environment?
To recycle receptors and have the most relevant ones on their membrane
90
What mediates the endocytotic pathways?
Clathrin and COP-I coated vesicles
(receptors remoulded and recycled by heat-shock proteins)
91
Where are proteins processed in plants?
The systema
92
How are organelles held in place in plant cells?
Tethering by the ER (uses specific accessory proteins -desmotubule, transvacuolar strand)
93
What do oil bodies do?
Spread and house triglycerides as a budding off of the ER
94
What are ER-tonoplast and ER-chloroplast said to be?
Semi-autonomous as they can grow and undergo fission without the cell cycle
BUT THEY CONTAIN DNA
95
What are microbodies?
Semi-autonomous organelles than house molecular components, BUT HAVE NO DNA
96
3 examples of microbodies and what they are?
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
What are plastids?
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
Examples of plastids?
Etioplast, chromoplast, chloroplast, leucoplast, amyloplast, elaioplast, proteinoplast
99
What does ___ contain:
- Embryo?
- Seed?
- Seedling?
- Mature leaf?
- Senescent leaf?
- 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
Why does primary cell wall contain cellulose synthases?
Produce macromolecular structures
101
Why does primary cell wall contain soluble proteins?
Destruction of bacteria
102
Why does primary cell wall contain cellulose microfibrils?
Long and provide core strength in protective matrix
103
What is the hemicellulose in primary cell wall?
Mesh-like polysaccharide proteins
104
What is the peltin in primary cell wall?
Gel-like inter-conecting polysaccharide galacturonans
105
Main role of primary cell wall?
Provides support and contains plasma membrane proteins
106
What is the difference between the secondary cell wall and primary?
Secondary has tighter arrangement of cellulose microfibrils and hemicellulose, also contains lignin which are water-impermeable polyphenolic molecules that add rigidity and support
107
Difference in cell wall between plants and fungi?
Plants = cellulose-pectin based
Fungi = chitin-based
108
Difference in junctions for adhesion and communication in plants and animals?
Plants = plasmodesmata (everything held in
cell wall)
Animals = tight+gap junctions, desmosomes
109
What are plasmodesmata for?
Osmotic control, communication and contribute to biomechanical sensing and signalling
110
What do plants have for organism flexibility?
Partially permeable primary cell walls stuck together with gel-like middle lamella
111
What are plasmodesmata?
Desmotubules that link to the ER with complex, branched architecture to maintain cell wall integrity
112
2 phases of plant life cycle?
Vegetative and reproductive
113
3 types of permanently vegetative cells/tissues?
1. Ground cells of the cortex
2. Vascular cells of vascular bundles
3. Epidermal/dermal cells of the dermal tissue
114
3 parts of dermal tissue?
Cuticle, trichomes (protect) and guard cells
115
3 types of ground/cortex tissues?
1. Parenchyma
2. Collenchyma
3. Sclerenchyma
116
Parenchyma:
- Level of specialisation?
- Role?
- Cell walls?
- Low
- Perform key metabolic functions
- Have thin and flexible primary, no secondary
117
Collenchyma:
- What are they?
- Cell walls?
- Differentiated parenchyma
- Thicker and more uneven primary, no secondary
118
Sclerenchyma:
- What are they?
- Cell walls?
- 2 types?
- 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
2 types of xylem cells?
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
What are sieve tube elements?
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
What happens when a signal molecule binds to a receptor?
Signalling downstream cascade in cell for response and activation of intracellular signalling molecules
122
What do cell surface receptors bind to?
Signal molecules that cannot pass through the plasma membrane
123
What do intracellular receptors bind to?
Small, hydrophobic signal molecules within the cell as they can pass through the plasma membrane
124
Difference between fast and slow responses?
Fast have rapid protein alterations to change function
Slow express new genes and form new proteins (alters protein synthesis)
125
What do phosphate kinases do?
Attach phosphate groups
126
What do phosphate phosphatases do?
Remove phosphate groups
127
How are GTP-binding proteins activated and inactivated?
Activated upon GTP binding
Inactivated upon GTPase activity as it hydrolyses GTP to GDP
128
How do GTP-binding proteins inactivate themselves?
Via intrinsic GTPase activity
129
What do trimeric GTP-binding (G) proteins do?
Relay signals from G-protein-coupled receptors
130
What do monomeric GTPases do?
Assist other cell surface receptors
131
Crucial role of G-protein coupled receptors (GPCRs)?
Transmitting signals from external environment to inside of cells
132
What physiological processes are GPCRs involved in?
Sensory perception, neurotransmission, hormone regulation, immune response
133
Types of signal molecules that bind to GPCRs?
Proteins, small peptides, derivatives of amino/fatty acids
134
Typical structure of GPCRs?
Have 7 hydrophobic transmembrane domains (single polypeptide chain that threads back and forth across lipid bilayer)
135
When are trimeric G proteins activated?
When an extracellular signal molecule binds a GPCR and it conformationally changes
136
General structure of trimeric G proteins?
Have alpha, beta and gamma protein subunits (2 of which are tethered to the plasma membrane by short lipid tails)
137
What are trimeric G proteins like in their resting state?
Tethered to inner leaflet of plasma membrane, alpha subunit is associated with a GDP molecule
138
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?
- 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
2 main roles of trimeric G proteins?
1. Directly regulate ion channels
2. Activate membrane-bound enzymes which produce small messenger molecules
140
What does the trimeric G protein regulation of ion channels cause?
Immediate change in state and behaviour of the cell
141
How is the heartbeat an example of trimeric G proteins regulating ion channels for a response?
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
Why are interactions between activated trimeric G proteins and enzymes slower and more complex?
As they lead to production of additional intracellular signalling molecules
143
What are small molecules generated by enzymes upon trimeric G protein binding called?
Second messengers (amplify and spread the intracellular signal)
144
What does trimeric G protein activation of adenylyl cyclase produce?
cAMP from ATP
145
What terminates cAMP signal?
Cyclic AMP phosphodiesterase converts cAMP to AMP
146
What do high levels of cAMP activate?
Cyclic-AMP-dependent protein kinase (PKA) as it changes conformation of regulatory protein that holds PKA and releases an active form of PKA
147
2 things activated PKA catalyses the phosphorylation of?
1. Cytoplasmic enzymes to change their activity
2. Transcription factors that can alter gene expression
148
How does adrenaline binding to a GPCR cause glycogen breakdown?
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
What is an example of trimeric G proteins directly regulating ion channels?
Vision (GPCRs called rhodopsin are utilised)
150
How does image processing in the eye work?
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
What are receptor-tyrosine kinases?
A type of enzyme-coupled receptor with a cytosolic domain with intrinsic kinase activity (phosphorylates amino acids)
152
What activates receptor-tyrosine kinases?
Ligand binding to extracellular domain
153
What do receptor-tyrosine kinases mediate?
Cell growth, survival and differentiation
154
RTK activation:
- What happens when ligand binds?
- What then happens?
- What does the phosphorylated tyrosine become?
- 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
3 main types of downstream RTK signalling?
1. Ras/MAPK pathway
2. PI3K-Akt pathway
3. Phospholipase C-gamma pathway
156
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?
- 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
MAP kinase cascade:
- What is it?
- What controls it?
- What is it an example of?
- 2 results to promote cell proliferation?
- A conformational signalling switch
- Phosphorylation status
- Amplification
- Protein phosphorylation or transcription factor phosphorylation
158
MAP kinase cascade:
- Protoncogenes?
- Concogenes?
- When are proteins active?
- Normal Ras and MAPK genes
- Constitutively activated versions of them, regardless of phosphorylation status
- When phosphorylated
159
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?
- 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
PLC pathway:
-What does activated RTK with PLC-gamma bound use PIP2 for?
- What is the fate of IP3?
- Role of DAG?
- 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
What do ligand-gated ion channels need to open?
Specific chemicals to bind
162
What are gated ion channels responsible for?
Signalling between nerve cells as they convert chemical signals to electrical signals
163
Role of voltage gated ion channels in nerve cell signalling?
Generate and carry action potential along axon to the presynaptic terminal
164
Role of ligand gated ion channels in nerve cell signalling?
Rapid transmission of signals across synapses in the nervous system via neurotransmitter binding to channel on postsynaptic membrane
165
Where is the acetylcholine receptor?
Between motor neuron and skeletal muscle cell
166
Glycine transmitter gated ion channel:
- What does it mediate?
- What does it process?
- When is it open?
- What happens when it is open?
- 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
What is necrosis?
Unplanned cell death that is usually in response to damage or infection (can cause further damage to surrounding cells and tissues)
168
How does apoptosis avoid issues associated wirh necrosis?
Dismantles cells
169
What occurs in S phase?
DNA replication
170
What occurs in G1 and G2 phases?
Growth and monitoring of internal+external environments to ensure conditions are suitable for replication
171
3 things a cell does during interphase?
1. Transcribes genes
2. Synthesises proteins
3. Grows in mass
172
3 cell cycle checkpoints?
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
When must DNA damage be fixed before?
DNA replication and the M phase
174
What regulates the start transition in late G1 phase?
Outside influences
175
What occurs when cells signal that more cells are needed?
Cell proliferation
176
Why can cancer result from cell cycle?
Malfunction in regulation of cell numbers can result in cancerous growth
177
CDKs:
- What part of cell cycle relies on them?
- Where are they present?
- Does concentration change?
- Role?
- How does activity rise and fall?
- Progression
- Dividing cells
- No
- Phosphorylate proteins in the cell cycle when activated (cyclin bound)
- In/activated by cyclin binding
178
Does cyclin change concentration through cell cycle and why is this important?
Yes as they drive cyclic assembly and activation of cyclin-CDK complexes
179
What do different cyclin-CDK complexes trigger?
Different steps in the cell cycle
180
Why does cyclin concentration change?
Rises are due to transcription of cyclin genes and formation of cyclin proteins, falls are due to degradation
181
What does cyclin degradation help with?
Driving entry into the next cell cycle phase
182
2 pathways for cells when they exit the cell cycle?
1. Undergo apoptosis
2. Enter G0
183
What are quiescent cells?
Cells withdrawn to G0, but can reenter the cell cycle under certain conditions
184
What is terminal differentiation?
Permanent withdrawal from the cell cycle (or in G0)
185
What is senescence?
Termination of cell division
186
What is immortalisation/transformation?
The evading of senescence cancer cells do
187
What is a benign tumour?
Excessive growth of cells in original tissue, less dangerous but can become malignant
188
What is a malignant tumour?
Cancerous, cells invade surrounding tissue
189
What are metastases?
Cancerous cells breaking down boundaries, entering the bloodstream to take over distant sites and form these metastases
190
2 contributors to cancer development?
1. Activation of proto-oncogene
2. Inactivation of a tumour suppressor gene
191
3 gene mutation pathways that can occur in individual tumours?
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
What do the proteins cyclin-CDK complexes phosphorylate play a role in?
Initiation of DNA replication, nuclear envelope breakdown, chromosome separation
193
What destroys cyclins?
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
How many times must DNA be replicated in cell division?
ONLY ONCE
195
What is the proliferating cell nuclear antigen (PCNA) part of?
DNA replication complex - PCNA is a sliding clamp
196
How is it ensured DNA only replicates once in the S phase?
Origins of replication only fire once and then the cyclin-CDK complexes are targeted for destruction by APC/C
197
What happens when APC/C is removed?
Origins of replication keep firing and the cells re-replicate the DNA but never divide
198
What do homologous chromosomes replicate to form?
Sister chromatids
199
What holds sister chromatids together until separation?
Cohesin rings
200
What are microtubules responsible for?
Polarising cell via forming bipoles (begins in S-phase with duplication of centrioles and centrosomes)
201
What is a centrosome?
Centrioles and a mass of proteins called the pericentriolar material
202
What is a centriole?
A microtubule array
203
3 steps of centriole duplication in S-phase?
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
Prophase:
- Role of condesins?
- How is mitotic spindle formed?
- Loop chromatin into tight bundles
- Centrosomes move apart
205
Prometaphase:
- What attaches to microtubules?
- How does the nuclear envelope break down?
- Condensed chromosomes
- Lamins are phosphorylated by cyclin B-CDK1 and the nuclear pore complexes disassemble
206
Metaphase:
- Where do sister chromatids line up?
- What is dynamic instability?
- What orientates mitotic spindle?
- Metaphase plate
- Microtubules grow slowly and shrink rapidly
- Microtubule physical force
207
Kinetochores:
- What are they?
- What do sensor proteins do?
- Protein complexes made of structural and signalling proteins that link chromatin and microtubles
- Monitor attachment of microtubules and sense tension on microtubules
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Spindle assembly checkpoint:
- What does it do?
- Role of APC/C?
- Role of separase?
- Inhibits APC/C when kinetochores are exposed
- Degrades securin
- Cleaves cohesin
209
What occurs in anaphase?
Cohesins holding together sister chromatids are degraded
210
Telophase:
- What do microtubules do?
- What begins to form on midline?
- Bundle and push newly formed nuclei apart
- A contractile ring to cleave the cells
211
What occurs in cytokinesis?
Contractile ring cinches and pinches, midbody forms at scission point
212
What forms when cytokinesis fails?
Binucleate cells
213
What does oocyte meiosis arrest in until ferilisation?
Metaphase II
214
How do homologous chromosomes pair?
Via complimentary DNA sequences
215
How do 2 pairs of sister chromatids swap genetic material?
Form a four-chromosome bivalent joined by synaptonemal complex (synapsis) that allows swapping
216
4 ways genetic diversification can occur?
1. Independent assortment
2. Crossing over (segment swapping)
3. Gene conversion (non-crossing over homologous recombination)
4. Non-disjunction (chromosome segregation going wrong)
217
Why does meiosis I only separate the homologous chromosomes?
Spindle body pulls one side of each chromosome so they stay together due to chiasma (but pairs split)
218
Why does meiosis II separate sister chromatids?
Spindle bodies pull either side
219
4 benefits of compartmentalised genetic material in eukaryotes?
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
What is the inner membrane of the nucleus continuous with?
The lumen of the ER
221
How did the nucleus evolve?
Nuclear DNA was attached to the membrane and then engulfed to form the nucleus
222
What are nuclear lamins?
Meshwork of filaments (related to intermediate filaments) on inner nuclear membrane that associate with DNA
223
How are nuclear lamins connected to the cytoskeleton?
By SUN/KASH proteins in inner membrane
By nesprins in outer membrane
224
What is chromatin?
DNA and RNA complex organised around nuclear matrix
225
Difference between euchromatin and heterochromatin?
Euchromatin is less visible, less dense and around the centre of the nucleus
Heterochromatin is darker, tightly packed and around lamina and nucleoli
226
What does discrete territories of chromosomes in nucleus allow?
Co-regulation of genes on different chromosomes (depending on orientation)
227
Why are Lamina-Associated Domains (LADs) rich in heterochromatin?
Due to the low level of transcription
228
What are nucleoli?
Non-membrane bound organelles in the nucleus
229
5 functions of nucleolus?
1. Transcription of rRNAs
2. Assembly of ribosomal subunits
3. Sensing and responding to stress
4. Cell cycle regulation
5. Cancer
230
What segments of chromosomes does the nucleolus contain?
1. Encode rRNA genes
2. Proteins for processing rRNAs
3. Ribosomal subunit proteins
4. Small nucleolar RNA oligonucleotides
231
3 other nuclear structures?
1. Cajal bodies
2. PML bodies
3. Speckles
232
What are cajal bodies?
Proteins that process RNA, organise the genome and have a dense foci of coilin protein
233
What PML bodies?
Proteins associated with cajal bodies and are involved in DNA repair, cell proliferation and apoptosis
234
What are speckles?
Proteins associated with gene transcription and mRNA processing (splicing)
235
What is exported out of the nucleus?
mRNA, rRNA, tRNA and assembled ribosomal units
236
What is imported into the nucleus from the cytoplasm?
RNA polymerase, ribosomal proteins, transcription factors, all other structural proteins
237
What are nuclear pore complexes?
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
What is required for the facilitated transport of mRNA into the nucleus?
ATP hydrolysis
239
What is required for the facilitated transport of proteins, tRNA and ribosomes into the nucleus?
GTP hydrolysis
240
What is required for the facilitated nuclear transport?
Nuclear transport receptors called karyopherins
241
How is mRNA exported out of the nucleus?
As a mRNA-protein (mRNP) complex that includes processing, capping and splicing proteins as well as export factors
242
Why does mRNA transport require ATP hydrolysis?
ATP hydrolysis releases Dbp5 in cytoplasm and triggers mRNP dissociation --> the energy is needed to maintain a transport gradient
243
What gradient is formed for protein import/export?
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
How do GTPases act as molecular switches?
Bind GTP and hydrolyse a phosphate group to form GDP (off)
245
What is GTP hydrolysis assisted by?
GAPs
246
What does GTP loading after hydrolysis?
GEFs
247
Role of importins?
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
Role of exportins?
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
What are nuclear localisation signals (NLSs)?
Protein motif anywhere on protein that binds to importins
250
What are nuclear export signals (NESs)?
Sequence with four hydrophobic amino acids that bind to exportins
251
How can NLSs and NESs be modified?
By phosphorylation
252
How does nucleocytoplasmic shuttling work?
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
How does a translocation event give rise to leukaemia?
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
Why do humans have 23 pairs of chromosomes but all other primates have 24?
Humans had a telomere-telomere fusion event
255
What shows DNA is not lost during development?
Animal cloning as transferred nucleus gives rise to whole organism (gene expression must be regulated and DNA can be reprogrammed following differentiation)
256
5 steps of animal cloning in sheep?
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
7 considerations when choosing an animal model?
1. Ease to maintain
2. Cost
3. Known genome sequence
4. Ease of breeding
5. Experimental advantages
6. Ethical considerations
7. Animal licence
258
3 advantages of an animal model when studying genetics?
1. Large number of offspring
2. Short gestation
3. Large array of mutants
259
3 advantages of an animal model when studying embryology?
1. Many embryos
2. External development
3. Robust but easy to manipulate embryos
260
3 advantages of an animal model when studying genomics?
1. Relevance to human genome
2. Disease models
3. Drug testing
261
7 steps of frog life cycle?
1. Gemetogenesis
2. Fertilisation
3. Cleavage
4. Gastrulation
5. Organogenesis
6. Larval stages
7. Maturity
262
How does cleavage form 8 blastomeres?
Central cleavage from animal to vegetal pole then right-angled cleavage and then perpendicular cleavage
263
What do fertilised frog eggs have?
A grey crescent opposite where sperm entered as cortical cytoplasm rotates 30 degrees
264
4 steps of gastrulation?
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
3 germ layers?
1. Ectoderm (outer)
2. Mesoderm (middle)
3. Endoderm (internal)
266
What is neuralation?
Notochord signals neural plate to form a groove that invaginates into a neural tube
267
What type of cleavage occurs in mammals, amphibians and insects?
Mammals = rotational
Amphibians = radial
Insects = superficial
268
- What is holoblastic cleavage?
- 5 types?
- Whole egg divided
- Radial, spiral, bilateral, rotational and radial
269
- What is meroblastic cleavage?
- 3 types?
- Part of egg divided (lots of yolk)
- Bilateral, discoidal, superficial
270
Where are cells smallest after cleavage in amphibians?
At the top
271
Where are there more blastomeres in amphibians?
Animal pole
272
How do mammal eggs cleave?
Asynchronistically and not at the same time, do not divide at 90 degrees (meridionally and equatorially instead)
273
How does superficial cleavage work in insects?
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
5 types of cell movements during gastrulation?
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
How do inductive signals work in patterning?
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
How does induction work?
Cell secretes an inducing signal, closest cells have high signal, further have low/no signal
277
How do transcription factors work?
Repress or activate expression of genes by binding to DNA elements
278
How do induction signals affect transcription factors?
Different factors affected by different signals (more signal = greater effect = different cell types)
279
How does blastula formation occur?
Through cleavage
280
What germ layer gives rise to the nervous system?
Ectoderm
281
What does cleavage immediately follow?
Fertilisation
282
What is mesoderm?
Germ layer that makes bones and muscle
283
What is a blastopore?
Structure that forms first embryo opening
284
What is neurulation?
Process that forms notochord and neural tube in Xenopus laevis embryos
285
What is induction?
Process where one group of cells/tissues influences development of neighbouring cells/tissues
286
What is forward genetics?
Starts with random mutation --> find phenotype --> find gene
287
What is reverse genetics?
Start with gene --> remove gene --> look at phenotype
288
Genetic screen:
- What are they?
- 3 steps?
- 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
1. Generating mutants
- Who are mutagenised?
- What then happens?
- What are the F1 crossed with?
- Why are the F2 generation then inbred?
- 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
2. What phenotypes are observed?
Loss-of/gain-of-function
291
3. Identify the gene:
- 6 steps?
- How is RNA location in the embryo identified?
- 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
- What is genetic engineering?
- 4 examples?
- A reverse genetics technique that modifies gene expression
- Transgenesis, targeted knockouts, CRISPR-Cas9, RNAi
293
Transgenesis:
- What is it?
- How does it work?
- 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
7 steps of targeted knockouts (removal of a gene)?
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
What are chimera?
Mixture of normal and targetted cells
296
What are heterozygous knockout offspring?
Offspring presenting recessive trait from ES cell donor
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