Midterm 2nd sem Flashcards
(198 cards)
1
Q
Cells in G0 phase
A
- Quiescent: cell withdraws from cell cycle, but can re-enter with stimulus
- Senescent: can not be directed to cell cycle, cell doesnt die it just doesnt divide
(if a senescent cell enters the cell cycle it can be pathological because we dont want it to divide for a reason)
2
Q
Restriction point
A
The deciding step if the cell commits to mitosis or not
(strength of mitogenic stim.)
- Early G1 phase
3
Q
G1/S checkpoint
A
Ensures cell has all necessary info for replication.
When cell moves past this point, replication must finish completely
4
Q
G2/M checkpoint
A
Checks DNA rep. is complete and that cell is large enough for mitosis to occur
5
Q
M/mitotic spindle checkpoint
A
Ensures that all spindles are properly attached to chromosomes before anaphase
6
Q
Is there a distinct border between S and G2?
A
No, S phase can continue till the end of G2
7
Q
Cell checkpoints
A
- Restriction point
- G1/S checkpoint
- G2/M checkpoint
- M/ms checkpoint
8
Q
What is a CDK?
A
Cyclin dependent Kinase
- Adds phosphate groups to proteins to activate/deactivate them
9
Q
What are cyclins?
A
Regulatory proteins that bind CDKs to activate them
10
Q
Early G1
(CDK & Cyclin)
A
- CDK 4/6
- Cyclin D
11
Q
Late G1 & G1/S
(CDK & Cyclin)
A
- CDK 2
- Cyclin E
12
Q
G1/S & S
(CDK & Cyclin)
A
- CDK 2
- Cyclin A
(SPF)
13
Q
G2/M & M
(CDK & Cyclin)
A
- CDK 1
- Cyclin B
(MPF)
14
Q
What CDK & Cyclin present in all phases of Cell cycle
A
- CDK 7
- Cyclin H
(CDK act. kinase)
15
Q
2 functions of CDK 7
A
1) Activates all CDKs in cell cycle
2) Transcription: Kinase activity in TFIIH complex phosph. RNA polymerase II, activated
16
Q
Negative effects on CDK activity
A
- CDK inhibitory proteins (CKI)
- Inhibitory phosphorylations (e.g. by Wee1)
17
Q
CDK7 - Cyclin H complex
A
CDK Activating Kinase
(CAK)
- Adds specific pi to thr residue on activation loop (T-loop)
18
Q
CKI
A
CDK Inhibitory Proteins
Bind CDK-Cyclin complex forming potential trimer
19
Q
4 conditions for CDK activation
A
- Appropriate Cyclin
- Phosphorylation by CAK on Thr
- No cyclin inh. protein (CKI)
- No Inh. phosphorylation (wee1) on Thr/Tyr
20
Q
INK4 Proteins
A
- Inhibits G1 phase by inhibiting CDK 4/6 complexes
- p16a / 15b / 18c / 19d
21
Q
CIP / KIP Proteins
A
- Inhibit the cell in response to trouble (e.g. DNA damage) during cycle
- Act on broader CDKs (4,6,2,1)
- p 21 / 27 / 57
22
Q
P21
A
- CIP protein
- Arrests cell in case of ‘trouble’ like DNA damage
23
Q
P27
A
- KIP protein
- Arrests cell in the S phase (2nd checkpoint- G1/S)
24
Q
CDC25
A
- Phosphatase enzyme
- Activates CDK-Cyclin complex by removing phosphates added on specific Thr/Tyr residues by Wee1
25
Transcriptional Repressor
Complex of E2F + DP + pRb (retinoblastoma)
Represses genes not needed for G0 and early G1
26
Transcriptional Activator
Transcriptional repressor complex after pRb was removed by CDK4,6-CyclinD dimer complex (protein kinase) phosphorylating pRb
Now cell must finish replication or die as it has passed the restriction point
27
S-phase Promoting Factor (SPF) & what keeps it inactive
CDK2-CyclinA complex
- p27, which is phosphorylated to remove and activate by CDK2-CyclinE
28
M-phase Promoting Factor (MPF)
CDK1-CyclinB complex
29
Wee1 & CDC25 relationship
Directly antagonistic
Wee1 is a kinase
CDC25 is a phosphatase
(both act on thr & tyr subunits)
30
Specific regulatory A.As on CDKs
- Threonine
- Tyrosine
31
MPF reactions
- Phosph. of cdc25 & Wee1
- Phosph. of Condensin (chromosomes)
- Phosph. of Lamin (nuclear env.)
- Phosph. of GM130 (division of Golgi & ER)
- Phosph. of Microtubule proteins MAPs (mitotic spindle)
- Phosph. of Myosin light chain to prevent immature cytokinesis
- Phosph. APC (anaphase prom. cplx) to degrade Cyclin B
32
How is MPF deactivated
MPF activates APC which tags Cyclin B for Ubiquitination which is then degraded by a proteasome
33
What happens if in M checkpoint 1 kinetochore is not connected
Mad & Bub proteins are activated which inhibit APC from starting anaphase
So the cell waits
When all connected Mad/Bub stop inh.
34
APC 2 big roles
1) Ubiq. Cyclin B = stops MPF
2) Ubiq. Securin which blocks Separase = Separase released = cuts cohesin = sisters chromatids separate (anaphase)
35
ATR
- Detects single ssDNA damage
- Serine/Threonine Kinase
- Phosph. CHK1 (activated)
36
ATM
- Detects dsDNA damage
- Serine/Threonine Kinase
- Phosph. CHK2 (activated)
37
ATR & ATM mechanism of action
1) CHK1/2 phosphorylated
2) CHK1/2 phosphorylate CDC25, inhibiting it
3) Wee1 in turn activated
38
p53 is known as
Number 1 Tumor supressor & it is a Transcription Factor
39
p53 structure
Transcription factor
- DNA binding domain
- Transactivation domain
- Tetramerization domain
- Proline rich domain
- C-terminal domain
- NLS/NES
40
Post-translational mods of p53
- Ubiquitination
- Acetyl-transferase
- Phosphorylation
41
p53 Ubiquitination
- Mostly on C-terminal domain
- Lysine residues ubiq.
- MDM2 is a specific ubiq. ligase & main inhibitor of p53
42
p53 Acetyl-transferase
- p300 recruited
- Acetylation of p53 histones
- More active
43
p53 Phosphorylation
Phosph. p53 if any problems:
- DNAPK (DNA breaks)
- ERK1/2 , JNK , p38 MAPK (stress)
- PKR (RNA virus replication)
- AMPK (energy deficiency)
- Phosph. can prevent MDM2 binding
(ALL ACTIVATE p53)
44
MDM2 actions
(binds where?)
- inhibits p53
- Binds TAD (transactivation d)
- Ubiq. CTD (c-term. d)
45
Activation of p53
1) p53 phosphorylated & enters nucleus
2) p53 bind p53RE
3) p300 recruited and acetylates histones
4) p53 activated more
46
Self-regulation of p53 (stress-free)
Lower p53 levels cause lower MDM2 levels, so overall p53 can start to rise a bit again
= controls p53 levels, not too high not too low
47
What links p53 with cell cycle regulation
p21 activation
Universal inhibitor of the cell cycle which is activated by p53 in case of any Damage
48
2 ways we can get Oncogenes
- Brought into the cell by a virus (DNA viruses or Acutely transforming Retroviruses)
- Formed within the cell
49
4 Factors regulating Tumorigenesis
- Cell Cycle activators/inhibitors
- Apoptosis activators/inhibitors
- GFs & their signaling
- Differentiation factors & signaling
50
Gadd45
Prevents dimerization of CDK1 & Cyclin B
- Activated by p53
51
14-3-3σ
Similar to GADD45 in effect
Grabs MPF (CDK1-CyclinB)
so stops from entering nucleus to trigger mitosis
52
ARF
Activates p53 by inhibiting MDM2
53
Protooncogenes def.
Genes involved in regulation of the cell cycle
If protooncogenes become faulty/mutated = Oncogenes
54
Protooncogene examples
- GFs
- Inhibitors of Apoptosis
- Cell cycle Activators
55
3 Main complexes that initiate Apoptosis
- Apoptosome
- DISC
- PIDDosome
56
What mediates Apoptosis
Intracellularly by Caspases
57
2 Apoptosis pathways
- Intrinsic / Mitochondrial pathway (apoptosome)
- Extrinsic pathway (DISC)
58
Apoptosome complex structure
- Cytochrome C
- Apaf1
- Procaspase-9
- CARD domains
59
Apoptosome activation
1) Cytochrome C released from mitochondria binds Apaf1
2) Apaf1 form large ring complex
3) CARD on Apaf1s recruite Procaspase-9
4) Apoptosome activated
60
dATP effect on Apoptosome
Binds Apaf1 helping to activate it
= Favors apoptosis
61
DISC complex structure
- Death receptor (Fas, TNF)
- FADD (Death & Death effector dom.)
- Procaspase 8/10
62
BID
Links Intrinsic and Extrinsic apoptotic pathways
63
BID pathway
1) BID to tBID by Caspase-8 from ext. pathway
2) tBID goes to mitochondria and reacts with pro-apoptotic protein BAX & BAK
3) Causes Cytochrome-C release from mitochondria
4) Intrinsic path. is also activated
(induced by p53)
64
PIDDosome complex structure
- 5 Death Domains (DDs)
- RAIDD (adaptor)
- Procaspase-2
- CARD domains
65
PIDDosome facts
- Newest, discovered a decade ago
- PIDD stands for p53 Induced protein with Death Domain
66
IAP
Inhibitor of Apoptosis protein
- To allow apoptosis, IAPs need to be inhibited
67
Apoptotic cell 'Eat Me' Signal
- Normally cells have Phosphatidylcholine on outside, and Phosphatidylserine inside by Translocase enzyme (flippase, floppase)
- In apoptosis, scramblase switches them so phosphatidylserine is on outisde
- Phagocytes eat the cell
68
Bcl2 family proteins
Tightly regulate the mitochondrial (intrinsic) apoptosis pathway
69
3 Classes of Bcl2 proteins
1) Proapoptotic multidomain Bcl2 proteins
2) Antiapoptotic multidomain Bcl2 proteins
3) Proapoptotic BH3 only Bcl2 proteins
70
Proapoptotic multidomain Bcl2 proteins
BAK & BAX
- Form a pore in outer membrane making mitochondria permeable to proteins
- Release of Cytochrome-C = intrinsic pathway
(induced by p53)
71
Anti-apoptotic multidomain Bcl2 proteins
Bcl2 / Bcl-X / MCL1
- Prevent pore formation on mitochondrial membrane (by blocking bak/bax)
72
Proapoptotic BH3 only Bcl2 proteins
BAD / BID / BIM / NOXA / PUMA
- Inhibit anti-apoptotic Bcl2 proteins
- Translocates BAX to mitochondria
(induced by p53)
73
Causes of Apoptosis
- Lack of survival signal: BAD protein
- Death ligand binding: tBID
- p53 activation: BID/PUMA/NOXA
- ER stress: BIM
74
Proteins released from intermembrane space
- Cytochrome C
- Smac & Omi (inhibit IAPs)
- AIF & EndoG (chromatin condensation & DNA fragmentation)
75
How survival signals work
- Inhibit BAD protein via phosphorylation (ser/thr) by PKB
- Active caspases can also be inh. by PKB phosphorylation
76
Caspases
- Endoproteases
- Cystine protease
- Asp cleavage site
- Cleave 'Death substrates'
77
Initiator & Effector Caspases
- Initiator: 8 / 9 / 10 (receive apoptotic signals)
- Effector: 3 / 6 / 7 (cut death substrates)
78
Caspase prodomains
Initiator prodomains are much larger and include DED (death effector d) & CARD (caspase recruitment d)
79
What cuts up DNA in Apoptosis?
CAD = Caspase Activated DNAase
- Cuts linker region of DNA into 180bp (or its multiples)
- Inhibited by ICAD
- Caspase-3 removes ICAD
80
Granzyme B info
- Protease enzyme
- Produced in NK cells & cytT
- Packed in vesicles with Perforin
81
Granzyme B mechanism
1) Cleaves pro-caspase-3
2) Active Caspase-3
3) can activate tBID to bring caspase-9 for further activation of procaspase-3
82
Caspase dependent cell death
- Apoptosis
- Pyroptosis
83
Caspase-independent cell death
- NETosis
- Parthanatos
- Ferroptosis
- Necroptosis
84
Pyroptosis
- Induced by infection (immune response to pathogen)
- Caspase-1 dependent (& 4, 5, 11)
85
Pattern Recognition Receptors (PRRs)
- Toll-like R (TLR) : Extracellular
- Nod-like R (NLR) : Intracellular
86
Pyroptosis Mechanism
1) Danger signal from Host/Pathogen
2) PRRs detect danger (TLR/NLR)
3) NLRP3 activates
4) Inflammosome formation
5) Caspase-1 activation (auto)
6) Caspase-1 cleaves & activates pro-IL-1B + pro-IL-18 to IL forms
7) Gasdermin D (GSMD) cleaved by C1 to N & C-term fragments
8) N-term fragments assemble pore
9) Irreversible chromatin condensation & lysis due to pore
87
Inflammosome components
- NLRP3
- ASC (+ CARD domain)
- Pro-Caspase-1
88
Role of Caspase-1 in Pyroptosis
- PM Pore formation
- Pro-inflamatory cytokines
- Chromosomal cleavage
89
Pyroptosis diseases examples
- Atherosclerosis
- Myocardial infarction
- Alzheimer's
- Parkinson's
- Ischemic stroke
- Tumors (double ended)
90
NETosis releases...
- Neutrophil chromatin
- Anti-pathogenic proteases
91
NETosis mechanism
1) Stimulus (pathogen)
2) ROS produced by Neut. my mitochondrial NADPH ox.
3) Azurophilic granule fusion (MPO, NE)
4) MPO activates NE
5) NE degrades actin fil. to enter Nucleus to cleave histones
6) Chromatin decondensation by Citrullation (PAD4+++) & Cleavage
7) DNA release by GSMD (NE breaks)
8) Extracellular DNA release
92
Enzymes in Azurophilic granules
Antimicrobial proteases & Enzymes
- Myeloperoxidase (MPO)
- Neutrophil Elastase (NE)
- Proteinase 3 (PR3)
- Cathepsin G
93
Myeloperoxidase role (MPO)
- Produces Hypochlorous acid (HClO) from H2O2 = anti-microbial
- Activated Neutrophil Elastase (NE)
94
How does chromatin decondense in NETosis
- Histone citrullation by PAD4+++ & deamination (Arg)
- Cleavage by Proteases
95
NETosis treatments
- DNase (break down NET)
- PAD4 inhibitors (prevent chromatin decondensation)
96
Parthanatos
- Caused by accumulation of Poly-ADP Ribose (PAR)
- Mitochondrial depletion of ADP & NAD+
97
Structure of PARP-1
- DNA binding domain (N-t & 2 zinc finger motifs)
- NLS
- Automodification domain
- Catalytic domain (NAD+ binding s)
98
PARP-1 function
- Adds poly-ADP-ribose chains on itself & target proteins in response to DNA damage (single/double)
- PAR chains are like magnets for DNA repair factors
99
PAR polymer metabolism
- PAR polymerase (PARP) adds PAR to proteins to signal DNA repair = cleaves NAD+ so it depletes
- PAR Glycohydrolase (PARG) cleaves the polymer from the target protein
100
Parthanatos Mechanism
1) PARP-1 recognizes DNA damage
2) (Hyper)Activation of PARP-1 due to DNA damage, parylation of itself & other proteins
3) NAD+ & ATP depletion causes bioenergetic collapse
4) PAR Glucohydrolas too overwhelmed
5) Mitochondrial accumulation of PAR & dysfunction = AIF release
6) AIF (guide) interacts with MIF (knife)
7) DNA degradation by MIF (DNA cleaving nuclease)
101
Parthanatos treatment
PARP-1 inhibition
102
Fenton reaction
H2O2 + Fe2+ = Fe3+ + HO* + OH-
103
Ferroptosis mechanism
1) Fenton reaction forms ROS (OH*)
2) PUFAs react with ROS = Lipid radical
3) Lipid radical + O2 = Lipid peroxyl Radical
4) LPR + PUFA = Lipid Peroxide!!! (toxic)
104
Ferroptosis counteracting mechs
- Glutathione Peroxidase (GPX4) converts Lipid peroxides to corresponding alcohols
- Glutathione Reductase (GSR) regens GSH
- Xc- system brings cyst in & glut out, Cys forms GSH
105
Ferroptosis treatment
Ferrostatin-1
Stops chain reaction of Lipid Peroxidation
106
Necroptosis
- Tumor
- Viral Infection
- When Apoptosis is Inh.
107
What forms pro-survival in Necroptosis
Complex 1
- TRADD
- TRAF2
- RIPK1
- cIAP 1/2
- FADD
108
Execution of Necroptosis
- RIPK1 not Ubiq.
- Caspase 8 inhibited
- RIPK1 bind RIPK3 (necrosome)
- RIPK3 phosph. MLKL
- MLKL is executioner, pore formation
109
Necroptosis & cancer
- Increase in Lung & Pancreatic cancers
- Decrease in Breast & Colorectal cancers
110
Necroptosis treatment
RIPK inhibitors
111
Apoptosis components induced by p53
- Death receptors
- Proapoptotic mulitdomain Bcl2 proteins BAX & BAK
- Proapoptotic BH3 only Bcl2 proteins NOXA & PUMA
- BID
- Apaf1
112
Proteostasis
"Protein homeostasis"
Ability of lining systems to maintain & regulate a balanced & functional proteome & maintain protein integrity
113
Protein structures
- Native: Functional, Biologically active
- Non-Native: Misfolded, denatured
114
How much of our Proteome is in Proteostasis network?
2000 out of 20,000 genes (10% of proteome)
- Protein synth. & folding: 400
- Maintenance of conf. stability: 300
- Protein degradation: >1000
115
Proteostasis Network
1) Biogenesis
2) Trafficking
3) Folding
4) Disposal
116
Types of Proteolysis
- Limited
- Complete
117
Limited Proteolysis
- Cleavage of specific peptide bonds
- Only a few sites cleaved
- Post-translational mod.
= New protein with altered function
118
Complete proteolysis
- Non-specific cleavage of peptide bonds at multiple sites
- Degradation of misfolded/damaged proteins
- Ubiq-Proteosomal system & Autophagy-Lysosome
= Oligopeptides & Amino acids
119
What enzymes carry out proteolytic cleavage
Protein Hydrolases
- Exopeptidases: N or C-term
- Endopeptidases: Internal peptides
120
Two-fold control
Controls Protein/Enzyme interaction
- Isolation of proteins in specific nano-compartments
- Chaperones & tags for interaction
121
Ubiquitin
- Glycine 76 residue (c-term)
- Isopeptide bond with target Lysine
- 4 genes code it: UBA 52/80, UBB, UBC
122
Polyubiquitination (& sites)
- Ubiquitin Conjugating Enzyme system
- Lys48: Proteosomal deg
- Lys63: Lysosomal deg
123
Ubiquitin Conjugating Enzyme system parts
- E1: Ubiquitin Activating Enzyme
- E2: Ubiquitin Conjugating Enzyme
- E3: Ubiquitin Ligase
124
Polyubiquitination Mechanism
1) Ubiq added to E1 by high E thioester bond (ATP)
2) E1 transfers ubiq to E2
3) E3 binds Target & E2
4) E3 channels ubiq to Amino NH2- group of target lysine side chain
(done at least 4 times)
125
Substrate recognition methods by E3
- Constitutive recognition
- Substrate modification
- E3 ligase modification
- Association of Ancillary protein (helper protein/adaptor)
126
Signals for Polyubiquitination
- N-end rule
- Hydrophobic patches
- PST sequences
127
Proteosome structure
- Regulatory particles (19s) x2
- Core particle (20s) x1
128
Proteosome Regulatory particles
- Lid: Closure & Deubiquitination
- Base: Ubiquitin-R & ATPase ring
129
Proteosome Core particle
- 2a rings (7su): Gating
- 2B rings (7su): Catalytic subunits
130
Proteosome core particle B catalysis
-B1: Caspase-like = - residues (asp, glu)
- B2: Trypsin-like = + residues (arg, lys)
- B5: Chymotrypsin-like = hydrophobic residues (phe, tyr)
131
Proteosome function
1) Binds to base of regulator part.
2) Lid opens
3) Engagement using ATP to fix
4) Conformational change for other RP to hide Ub-R
5) De-ubiquinating enzyme
6) Unfolding by ATPase and translocation to core CP
7) a-ring opens, B catalysis
Oligopeptide release
132
Proteosome inhibitor
Bortezomib
133
What regulates Proteosome expression
NRF1
- Normally degraded
- When we have prototoxic stress NRF1 not degraded, genes are activated to make proteosomes & deubiquitinase removes ubiq.
134
Immunoproteosome
- Similar structure to proteosome
- Highly abundant in APCs
- Degrades proteins for antigen presentation on MHC-1
- Oligopeptides by TAP to ER, then to MHC1 through Golgi
135
Immunoproteosome structure
- 11s subunit (PA28) (ATP independent)
- B subunits: B1i, B2i, B5i
136
Immunoproteosome Function
- Proteosomal degredation
- TAP (transporter for antigen processing)
137
TAP
- (immuno)Proteosome cuts proteins to peptides
- TAP transports peptides to ER
- Loaded on MHC-1 molecules
- Alert cytotoxic T cells
138
Why are proteins aggregates dangerous
Form B-sheet structures with hydrophobic backbone aggregating at hydrophobic environments
e.g membranes forming pores, TFs or chaperones stopping them from carrying out their job
139
Aggresome formation
1) Misfolded proteins accumulate
2) Parkin/ubiq ligase ubiqs at Lys63
3) Further aggregation
4) Move along microtubules juxtanuclearly
5) Autophagosome forms, fuses with Lysosome
140
Lysosome
- Primary or Secondary (new/fused)
- Acidic pH 4-5
- Single bilayer
- V-type ATPase (pump protons in)
- Permease to pump out degraded products for recycling
141
Types of Autophagy
- Macroautophagy
- Microautophagy
- Chaperone-mediated Autophagy (CMA)
142
Macroautophagy & steps
- Breakdown of bulk cellular components
- Activated in early fasting (30m - 8h)
- Initiation, Nucleation, Elongation, Closure, Transport (fusion with lysosome)
143
Macroautophagy Mechanism
1) Initiation, ULK1 dephosph. activated when mTOR inhibited
2) Nucleation, ULK1 phosph. Beclin1-Vps34 = PI3K leads to PI3P
3) Elongation, PI3P recruits proteins to expand phagopore like LC3 to anchor (phosphatidylethanolamin)
4) Closure, ESCRT-III helps close membrane
5) Transport & Fusion, Autophagosome moves to Lysosome by Dyenin guided by Rab7 for docking, tags cargo by p62, SNARES to fuse
144
What is ULK-1, what activates and inhibits it
Serine/Threonine Kinase essential for autophagy initiation
- mTOR inh
- AMPK act.
145
Regulation of Macroautophagy
- mTOR/AMPK balance
- TFEB (transcriptional reg.)
146
TFEB
Transcription factor activating genes involved in autophagy
(inh by mTOR)
147
Microautophagy
- Breakdown by direct engulfment of cytoplasmic material
- Non-selective
- Invagination, Sequestration, Fusion, Degredation
148
Chaperone Mediated Autophagy (CMA)
- Very Selective, Receptor mediated protein & lipid droplet breakdown
- Only soluble cytosolic proteins with a KFERQ-like motif are eligible
- LAMP-2A receptor for this
- In Hypoxia, late starvation (10-30h), Oxidative stress
149
CMA steps
1) Cytosolic HSC70 binds proteins with KFERQ motif
2) CMA complex formed
3) CMA comp. binds LAMP-2A on surface & oligomerizes to channel
4) Protein unfolded to fit through LAMP-2A channel (stabilized by GFAP)
5) Lysosomal lys-HSC70 helps pull protein in (ATP)
150
Rapamycin
mTOR inhibitor which activates autophagy
- Increases life-span of mice when tested
151
How can viruses minimize size of Nucleic acids?
- Overlapping genes
- Frame shifting
- Gene can be used in both directions
152
Two ways Bacteriophages replicate
- Lytic Cycle
- Lysogenic Cycle
153
Lytic Cycle
- Viral DNA enters 'Phage receptor'
- Host cell DNA replication blocked & degraded
- Viral DNA replicated and new progeny phages assembled inside cell
- Lysis and spread of new phages
~300 in 22 minutes
154
DNA made in Lytic cycle called
Rolling circle DNA replication
= Concatemers
155
Lytic cycle Genome packing
Terminase enzyme grabs concatemer and directed to empty procaspid (portal)
156
How host can distinguish own vs invader DNA
- EcoR1 (rest. endonuc) cuts DNA sequences not methylated on both ends
- SAM methyl group donor
- Phage DNA contains 5-HMC / Glucosyl-HMC which misleads EcoR1
157
What phages undergo Lytic cycle
T-phages (T2/4/6) T4!
Lambda Phages
Infect E.coli
158
What phages undergo Lysogenic cycle
Only Lambda phages (& other temperate phages)
159
Lysogenic Cycle
- Phage DNA not replicated but Integrated to host DNA = prophage
- Bacteria carries prophage = Lysogen
- Phage DNA stays silent except for Lambda repressor (C1)
160
How is DNA integrated in Lysogenic Cycle
- attP & attB match
- Viral integrase + Integration host factor (IHF) needed
- Excisionase needed when it wants to switch to lytic
161
SOS viral response
1) DNA stress causes RecA protein activation
2) RecA breaks bacterial repressor LexA & C1 Lambda repressor
3) Repair turned on
4) Cro gene expressed blocks C1 more
5) Full phage replication= Lytic cycle
162
What determines if Lytic or Lysogenic Cycle happens
- C2: Makes C1 lambda repressor= Lysogenic
- Cro: Blocks C1 lambda repressor= Lytic
163
Virion
Only refers to the complete infectious particle within the Virus able to infect the host
164
Baltimore System
Groups viruses according to their type of Genetic material & how it is used to make mRNA
165
Reverse transcriptase activities
- RNA dependent DNA polymerase
- 3' Ribonuclease
- DNA dependent DNA polymerase
166
HIV capsid contains
- 2x + ssRNA
- Reverse transcriptase
- tRNA (lysine) as primer for RTase
- Integrase
167
3 Major genes in Retroviral Genome
(& what for)
Gag, Pol, Env
- Gag: codes for Capsid proteins
- Pol: codes for RTase, Integrase, Protease
- Env: codes for Envelope proteins
168
Why are LTRs essential for Viruses
For entry into the nucleus & integration into host DNA & Transcription because it is a Promoter for viral DNA
169
Replication of HIV steps
1) Attachment & Fusion/entry
2) Reverse Transcription (cyt)
3) Integration (nucleus)
4) Transcription, Replication
5) Assembly
6) Budding & Maturation
170
Interferons types & origin
- INFa: Leukocytes
- INFB: Fibroblasts
171
What do IFNs induce production of
- Oligo A (oligoadenylate synthase)
- Ribonuclease L
- dsRNA dependent protein kinase (PKR)
172
In-Vivo Gene therapy
- Genes transferred into cells in the patient when cells cant be cultured in-vitro (e.g. brain)
- e.g. Cystic fibrosis, Spinal muscular atrophy (treated with adeno-associated viral vector)
173
Ex-Vivo Gene therapy
- Cells isolated from patient and cultured with therapeutic genes
- Modified cells selected and transferred to patient
- 1st used for Adenosine deaminase def. which accumulated deoxy-aminase = destroys T-lymphocytes
174
Viral Vectors & types
Viruses deliver new gene by infecting the cell but modified to not cause disease
- Retrovirus (ssRNA int.)
- Adenovirus (dsDNA, imm.)
- Adeno-associated Virus (ssDNA, int.)
- Herpes Sumplex Virus based (dsDNA)
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Non-viral vector types
- Pure DNA construct
- Lipoplexes
- Polyplex
- Human artifical chromosome
- DNA molecular conjugates
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Therapeutic strategies in Gene therapy
- Gene Augmentation
- Direct/Indirect cell killing
- Targeted inhibition
- Targeted gene mutation correction
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Gene Augmentation
- Diseases where gene lost function, we increase the amount of normal gene product to a level where the normal phenotype is restored
- Most common gene therapy
- Only when pathogenesis is reversible (autosomal recessive)
e.g. CFTR in Cystic fibrosis
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CRISPR & Cas9 stands for
- CRISPR: Clustered Regularly Interspersed Short Palindromic Repeat
- Cas9: CRISPR associated nuclease 9
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CRISPR/Cas9
Used in Bacterial immune system (adaptive) granting resistance against bacteriophages
1st discovered in E.coli
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CRISPR structure
- Short palindromic repeats (20-50)
- Identical but interspaced
- Unique Spacers between repeats
- Spacers found to be viral/bacteriophage DNA
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CRISPR associated genes
(cas genes)
- Helicases (unwind)
- Nucleases (cut)
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CRISPR 1st Viral infection
Immunization
1) Viral DNA injected
2) Cas1 & Cas2 recognize viral DNA
3) Cas1/2 cut near PAM sequence
4) Cut piece/protospacer added as a spacer on CRISPR locus bw repeats
5) Memory created
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CRISPR 2nd Viral infection
1) CRISPR region transcribed to pre-crRNA
2) tracrRNA binds complementary repeat sequences (duplex)
3) Duplex cleaved by RNase III cut into tracrRNA + crRNA hybrids (spacer, repeat, tracrRNA)
4) Hybrid binds Cas9 endonuclease to form CRISPR surveillance complex (Cas9 + crRNA + tracrRNA)
5) If same sequence encountered, Cas9 cuts it stopping the infection
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CRISPR/Cas9 in Biotechnology
1) crRNA & tracrRNA fused to form sgRNA (single guide)
2) sgRNA is complementary to specific 20-nt sequence in target DNA
3) tDNA followed by PAM having NGG sequence
4) Cas9 causes break in dsDNA 3bp upstream of PAM
5) Endogenous DNA repair mech.
- Non homologous end joining (ruins)
- Homology directed repair (fixes)
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Transgenic animal roles
- Disease models (oncomouse)
- Transpharmers (protein in milk)
- Xenoplanters (organ trans.)
- Food sources
- Scientific models
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Ways of delivery of genetic modification
- Microinjection (into zygote)
- Transfection (embryonic SC + vector)
- Electroportation
- Retroviral Vectors
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Knock-in technique
Insertion of a transgene or modified allele producing a gain of function mutation
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Knock-out technique
Removal / inactivation of a gene to test its function (based on homologous combination where working gene is swapped with another)
- Gene vector in Embryonic SC inserting NeoR-gene & injecting into blastocyst forming chimeric mice for breeding
- e.g. Neomycin-resistance element (neo-r gene)
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Conditional Knock-out technique
When we want to selectively knock-out a gene in a specific tissue/cell type or a particular developmental stage
GFP (green fluroesc. p) used as a signal for Cre & LoxP
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Knock-down technique
Partially or temporarily reducing a gene's expression
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Methods of mRNA degradation in Knock-down technique
- RNA interference (RNAi)
- Hammerhead Ribozyme
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RNA interference Knock-down (RNAi)
1) dsRNA cut into siRNA by Dicer
2) siRNA binds host RISC complex
3) RISC uses siRNA to find complementary mRNA
4) RISC cuts mRNA using Ago & its degraded so protein is no longer made
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Hammerhead Ribozyme Knock-down
1) Design hammerhead ribozyme complementary to mRNA
2) Transfect ribozyme to cytosol
3) Ribozyme binds & cleaves target mRNA leading to gene knock-down
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Directed Genome editing methods
- CRISPR/Cas9
- TALENs
- Zinc Finger Nucleases (like talens)
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TALENs
Transcription activator-like Effector Nucleases
- DNA binding domain & Nuclease domain
- Knicks dsDNA to promote repair
(zinc finger nucleases work the same)
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Cloning method
Somatic Cell Nuclear transfer
1) Enucleate a fertilized egg
2) Take nucleus from adult somatic cell
3) Fuse enucleated egg with somatic nucleus using Electric shock
4) Implant to surrogate
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Cloning types
- Reproductive cloning: Create living organism
- Therapeutic cloning: Stem cells for treatment
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Bond bw Ubiq. and Lysine
Isopeptide Bond
