Midterm 2nd sem Flashcards
1
Q
Restriction point
A
The deciding step if the cell commits to mitosis or not
(strength of mitogenic stim.)
- In G1 phase
2
Q
G1/S checkpoint
A
Ensures cell has all necessary info for replication.
When cell moves past this point, replication must finish completely
3
Q
G2/M checkpoint
A
Checks DNA rep. is complete and that cell is large enough for mitosis to occur
4
Q
M/mitotic spindle checkpoint
A
Ensures that all spindles are properly attached to chromosomes before anaphase
5
Q
Is there a distinct border between S and G2?
A
No, S phase can continue till the end of G2
6
Q
Cell checkpoints
A
- Restriction point
- G1/S checkpoint
- G2/M checkpoint
- M/ms checkpoint
7
Q
What is a CDK?
A
Cyclin dependent Kinase
- Adds phosphate groups to proteins to activate/deactivate them
8
Q
What are cyclins?
A
Regulatory proteins that bind CDKs to activate them
9
Q
Early G1
(CDK & Cyclin)
A
- CDK 4/6
- Cyclin D
10
Q
Late G1 & G1/S
(CDK & Cyclin)
A
- CDK 2
- Cyclin E
11
Q
G1/S & S
(CDK & Cyclin)
A
- CDK 2
- Cyclin A
(SPF)
12
Q
G2/M & M
(CDK & Cyclin)
A
- CDK 1
- Cyclin B
(MPF)
13
Q
What CDK & Cyclin present in all phases of Cell cycle
A
- CDK 7
- Cyclin H
(CDK act. kinase)
14
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
15
Q
Negative effects on CDK activity
A
- CDK inhibitory proteins
- Inhibitory phosphorylations (e.g. by Wee1)
16
Q
CDK7 - Cyclin H complex
A
CDK Activating Kinase
(CAK)
- Adds specific pi to thr residue on activation loop (T-loop)
17
Q
CKI
A
CDK Inhibitory Proteins
Bind CDK-Cyclin complex forming potential trimer
18
Q
4 conditions for CDK activation
A
- Appropriate Cyclin
- Phosphorylation by CAK
- No cyclin inh. protein (CKI)
- No Inh. phosphorylation (wee1)
19
Q
INK4 Proteins
A
- Inhibits G1 phase by inhibiting CDK 4/6 complexes
- p16a / 15b / 18c / 19d
20
Q
CIP / KIP Proteins
A
- Inhibit the cell in response to trouble (e.g. DNA damage)
- Act on broader CDKs (4,6,2,1)
- p 21 / 27 / 57
21
Q
P21
A
- CIP protein
- Arrests cell in case of ‘trouble’ like DNA damage
22
Q
P27
A
- KIP protein
- Arrests cell in the S phase (2nd checkpoint)
23
Q
CDC25
A
- Phosphatase enzyme
- Activates CDK-Cyclin complex by removing phosphates added on specific residues by Wee1
24
Q
Transcriptional Repressor
A
Complex of E2F + DP + pRb (retinoblastoma)
Represses genes not needed for G0 and early G1
25
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
26
S-phase Promoting Factor (SPF) & what keeps it inactive
CDK2-CyclinA complex
- p27, which is phosphorylated to remove and activate by CDK2-CyclinE
27
M-phase Promoting Factor (MPF)
CDK1-CyclinB complex
28
Wee1 & CDC25 relationship
Directly antagonistic
Wee1 is a kinase
CDC25 is a phosphatase
(both act on thr & tyr subunits)
29
Specific regulatory A.As on CDKs
- Threonine
- Tyrosine
30
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
31
How is MPF deactivated
MPF activates APC which tags Cyclin B for Ubiquitination which is then degraded by a proteasome
32
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.
33
APC 2 big roles
1) Ubiq. Cyclin B = stops MPF
2) Ubiq. Securin which blocks Separase = Separase released = cuts cohesin = sisters chromatids separate (anaphase)
34
ATR
- Detects single ssDNA damage
- Serine/Threonine Kinase
- Phosph. CHK1 (activated)
35
ATM
- Detects dsDNA damage
- Serine/Threonine Kinase
- Phosph. CHK2
36
ATR & ATM mechanism of action
1) CHK1/2 phosphorylated
2) CHK1/2 phosphorylate CDC25
3) Wee1 in turn activated
37
p53 is known as
Number 1 Tumor supressor
38
p53 structure
Transcription factor
- DNA binding domain
- Transactivation domain
- Tetramerization domain
- Proline rich domain
- C-terminal domain
- NLS/NES
39
Post-translational mods of p53
- Ubiquitination
- Acetyl-transferase
- Phosphorylation
40
p53 Ubiquitination
- Mostly on C-terminal domain
- Lysine residues ubiq.
- MDM2 is a specific ubiq. ligase & inhibitor of p53
41
p53 Acetyl-transferase
- p300 recruited
- Acetylation of p53 histones
- More active
42
p53 Phosphorylation
Phosph. p53 if any problems:
- DNAPK (DNA breaks)
- ERK1/2 , JNK , p38 MAPK (stress)
- PKR (RNA virus replication)
- AMPK (energy deficiency)
(ALL ACTIVATE p53)
43
MDM2 actions
- inhibits p53
- Binds TAD
- Ubiq. CTD
44
Activation of p53
1) p53 phosphorylated & enters nucleus
2) p53 bind p53RE
3) p300 recruited and acetylates histones
4) p53 activated more
45
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
46
Gadd45
Prevents dimerization of CDK1 & Cyclin B
- Activated by p53
47
14-3-3σ
Similar to GADD45 in effect
Grabs MPF (CDK1-CyclinB)
so stops from entering nucleus to trigger mitosis
48
ARF
Activates p53 by inhibiting MDM2
49
Protooncogenes def.
Genes involved in regulation of the cell cycle
If protooncogenes become faulty/mutated = oncogenes
50
Protooncogene examples
- GFs
- Inhibitors of Apoptosis
- Cell cycle Activators
51
What viruses can cause cancer
- DNA viruses (benign tumor)
- Acutely-transforming Retroviruses
52
3 Main complexes that initiate Apoptosis
- Apoptosome
- DISC
- PIDDosome
53
What mediates Apoptosis
Intracellularly by Caspases
54
2 Apoptosis pathways
- Intrinsic / Mitochondrial pathway (apoptosome)
- Extrinsic pathway (DISC)
55
Apoptosome complex structure
- Cytochrome C
- Apaf1
- Procaspase-9
- CARD
56
Apoptosome activation
1) Cytochrome C from mitochondria binds Apaf1
2) Apaf1 form large ring complex
3) CARD on Apaf1s recruite Procaspase-9
4) Apoptosome activated
57
dATP effect on Apoptosome
Binds Apaf1 helping to activate it
= Favors apoptosis
58
DISC complex structure
- Death receptor (Fas, TNF)
- FADD (Death & Death effector dom.)
- Procaspase 8/10
59
BID
Links Intrinsic and Extrinsic apoptotic pathways
60
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)
61
PIDDosome complex structure
- 5 Death Domains (DDs)
- RAIDD
- Procaspase-2
62
PIDDosome facts
- Newest, discovered a decade ago
- PIDD stands for p53 Induced protein with Death Domain
63
IAP
Inhibitor of Apoptosis protein
- To allow apoptosis, IAPs need to be inhibited
64
Apoptotic cell 'Eat Me' Signal
- Normally cells have Phosphatidylcholine on outside, and Phosphatidylserine inside by Translocase enzyme
- In apoptosis, scramblase switches them so phosphatidylserine is on outisde
- Phagocytes eat the cell
65
Bcl2 family proteins
Tightly regulate the mitochondrial (intrinsic) apoptosis pathway
66
3 Classes of Bcl2 proteins
1) Proapoptotic multidomain Bcl2 proteins
2) Antiapoptotic multidomain Bcl2 proteins
3) Proapoptotic BH3 only Bcl2 proteins
67
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)
68
Anti-apoptotic multidomain Bcl2 proteins
Bcl2 / Bcl-X / MCL1
- Prevent pore formation on mitochondrial membrane
69
Proapoptotic BH3 only Bcl2 proteins
BAD / BID / BIM / NOXA / PUMA
- Inhibit anti-apoptotic Bcl2 proteins
- Translocates BAX to mitochondria
(induced by p53)
70
Causes of Apoptosis
- Lack of survival signal: BAD protein
- Death ligand binding: tBID
- p53 activation: BID/PUMA/NOXA
- ER stress: BIM
71
Proteins released from intermembrane space
- Cytochrome C
- Smac & Omi (inhibit IAPs)
- AIF & EndoG (dismantle DNA)
72
How survival signals work
- Inhibit BAD protein via phosphorylation (ser/thr) by PKB
- Active caspases can also be inh. by PKB phosphorylation
73
Caspases
- Endoproteases
- Cystine protease
- Asp cleavage site
- Cleave 'Death substrates'
74
Initiator & Effector Caspases
- Initiator: 8 / 9 / 10
- Effector: 3 / 6 / 7
75
Caspase prodomains
Initiator prodomains are much larger and include DED & CARD
76
Granzyme B info
- Protease enzyme
- Produced in NK cells & cytT
- Packed in vesicles with Perforin
77
Granzyme B mechanism
1) Cleaves pro-caspase-3
2) Active Caspase-3
3) can activate tBID to bring caspase-9 for further activation
78
Caspase dependent cell death
- Apoptosis
- Pyroptosis
79
Caspase-independent cell death
- NETosis
- Parthanatos
- Ferroptosis
- Necroptosis
80
Pyroptosis
- Induced by infection (immune response to pathogen)
- Caspase-1 dependent (& 4, 5, 11)
81
Pattern Recognition Receptors (PRRs)
- Toll-like R (TLR) : Extracellular
- Nod-like R (NLR) : Intracellular
82
Inflammosome components
- NLRP3
- ASC
- Pro-Caspase-1
83
Role of Caspase-1 in Pyroptosis
- PM Pore formation
- Pro-inflamatory cytokines
- Chromosomal cleavage
84
Pyroptosis diseases examples
- Atherosclerosis
- Myocardial infarction
- Alzheimer's
- Parkinson's
- Ischemic stroke
- Tumors (double ended)
85
NETosis
- Neutrophil chromatin
- Anti-pathogenic proteases
86
Enzymes in Azurophilic granules
- Myeloperoxidase (MPO)
- Neutrophil Elastase (NE)
- Proteinase 3 (PR3)
- Cathepsin G
87
Myeloperoxidase role (MPO)
- Produces Hypochlorous acid (HClO) from H2O2 = anti-microbial
- Activated Neutrophil Elastase (NE)
88
How does chromatin decondense in NETosis
- Histone citrullation by PAD4 & deamination
- Cleavage by Proteases
89
NETosis treatments
- DNase (break down NET)
- PAD4 inhibitors (prevent chromatin decondensation)
90
Parthanatos
- Caused by accumulation of Poly-ADP Ribose (PAR)
- Mitochondrial depletion of ADP & NAD+
91
Structure of PARP-1
- DNA binding domain (N-t & 2 zinc finger motifs)
- NLS
- Automodification domain
- Catalytic domain (NAD+ binding s)
92
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
93
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
94
Parthanatos treatment
PARP-1 inhibition
95
Fenton reaction
H2O2 + Fe2+ = Fe3+ + HO* + OH-
96
Ferroptosis treatment
Ferrostatin-1
Stops chain reaction of Lipid Peroxidation
97
Necroptosis
- Tumor
- Viral Infection
- When Apoptosis is Inh.
98
What forms pro-survival in Necroptosis
Complex 1
- TRADD
- TRAF2
- RIPK1
- cIAP 1/2
- FADD
99
Execution of Necroptosis
- RIPK1 not Ubiq.
- Caspase 8 inhibited
- RIPK1 bind RIPK3 (necrosome)
- RIPK3 phosph. MLKL
- MLKL is executioner, pore form.
100
Necroptosis & cancer
- Increase in Lung & Pancreatic cancers
- Decrease in Breast & Colorectal cancers
101
Necroptosis treatment
RIPK inhibitors
102
Apoptosis components induced by p53
- Death receptors
- Proapoptotic mulitdomain Bcl2 proteins BAX & BAK
- Proapoptotic BH3 only Bcl2 proteins NOXA & PUMA
- BID
- Apaf1
103
Proteostasis
"Protein homeostasis"
Ability of lining systems to maintain & regulate a balanced & functional proteome & maintain protein integrity
104
Protein structures
- Native: Functional, Biologically active
- Non-Native: Misfolded, denatured
105
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
106
Proteostasis Network
1) Biogenesis
2) Trafficking
3) Folding
4) Disposal
107
Types of Proteolysis
- Limited
- Complete
108
Limited Proteolysis
- Cleavage of specific peptide bonds
- Only a few sites cleaved
- Post-translational mod.
= New protein with altered function
109
Complete proteolysis
- Non-specific cleavage of peptide bonds at multiple sites
- Degradation of misfolded/damaged proteins
- Ubiq-Proteosomal system & Autophagy-Lysosome
= Oligopeptides & Amino acids
110
What enzymes carry out proteolytic cleavage
Protein Hydrolases
- Exopeptidases: N or C-term
- Endopeptidases: Internal peptides
111
Two-fold control
Controls Protein/Enzyme interaction
- Isolation of proteins in specific nano-compartments
- Chaperones & tags for interaction
112
Ubiquitin
- Glycine 76 residue (c-term)
- Isopeptide bond with target Lysine
- 4 genes code it: UBA 52/80, UBB, UBC
113
Polyubiquitination
- Ubiquitin Conjugating Enzyme system
- Lys48: Proteosomal deg
- Lys63: Lysosomal deg
114
Ubiquitin Conjugating Enzyme system parts
- E1: Ubiquitin Activating Enzyme
- E2: Ubiquitin Conjugating Enzyme
- E3: Ubiquitin Ligase
115
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 NH2- group of target lysine side chain
(done at least 4 times)
116
Substrate recognition methods by E3
- Constitutive recognition
- Substrate modification
- E3 ligase modification
- Association of Ancillary protein (helper protein)
117
Signals for Polyubiquitination
- N-end rule
- Hydrophobic patches
- PST sequences
118
Proteosome structure
- Regulatory particles (19s) x2
- Core particle (20s) x1
119
Proteosome Regulatory particles
- Lid: Closure & Deubiquitination
- Base: Ubiquitin-R & ATPase ring
120
Proteosome Core particle
- 2a rings (7su): Gating
- 2B rings (7su): Catalytic subunits
121
Proteosome core particle B catalysis
- B1: Caspase-like = - residues (asp, glu)
- B2: Trypsin-like = + residues (arg, lys)
- B3: Chymotrypsin-like = hydrophobic residues (phe, tyr)
122
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
123
Proteosome inhibitor
Bortezomib
124
What regulates Proteosome expression
NRF1
- Normally degraded
- When we have prototoxic stress NRF1 not degraded, genes are activated to make proteosomes
125
Immunoproteosome
- Similar structure to proteosome
- Highly abundant in APCs
- Degrades proteins for antigen presentation on MHC-1
126
Immunoproteosome structure
- 11s subunit (PA28) (ATP independent)
- B subunits: B1i, B2i, B5i
127
Immunoproteosome Function
- Proteosomal degredation
- TAP (transporter for antigen processing)
128
TAP
- (immuno)Proteosome cuts proteins to peptides
- TAP transports peptides to ER
- Loaded on MHC-1 molecules
- Alert cytotoxic T cells
129
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
130
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
131
Lysosome
- Primary or Secondary (new/fused)
- Acidic pH 4-5
- Single bilayer
- V-type ATPase
- Permease to pump out degraded products for recycling
132
Types of Autophagy
- Macroautophagy
- Microautophagy
- Chaperone-mediated Autophage (CMA)
133
Macroautophagy & steps
- Breakdown of bulk cellular components
- Activated in early fasting (30m - 8h)
- Initiation, Nucleation, Elongation, Closure, Transport
134
What activated and inhibits ULK-1
- mTOR inh
- AMPK act.
135
TFEB
Transcription factor activating genes involved in autophagy
(inh by mTOR)
136
Microautophagy
- Breakdown by direct engulfment of cytoplasmic material
- Non-selective
- Invagination, Sequestration, Fusion, Degredation
137
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
138
CMA steps
1) Cytosolic HSP70 binds proteins with KFERQ motif
2) CMA complex formed
3) CMA comp. binds LAMP-2A and oligomerizes
4) Protein unfolded to fit through LAMP-2A channel
5) Lys-HSC70 helps pull protein in (ATP)
139
How can viruses minimize size of Nucleic acids?
- Overlapping genes
- Frame shifting
- Gene can be used in both directions
140
Two ways bacteria replicate
- Lytic Cycle
- Lysogenic Cycle
141
Lytic Cycle
- Viral DNA enters 'Phage receptor'
- Host cell DNA replication blocked
- Viral DNA replicated and new phages assembled inside cell
- Lysis and spread of new phages
~300 in 22 minutes
142
DNA made in Lytic cycle called
Rolling circle DNA replication
= Concatemers
143
Lytic cycle Genome packing
Terminase enzyme grabs concatemer and directed to empty procaspid
144
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
145
What phages undergo Lytic cycle
T-phages (T2/4/6) T4!
Lambda Phages
Infect E.coli
146
What phages undergo Lysogenic cycle
Only Lambda phages (& other temperate phages)
147
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)
148
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
149
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
150
What determines if Lytic or Lysogenic Cycle happens
- CII: Makes C1 lambda repressor= Lysogenic
- Cro: Blocks C1 lambda repressor= Lytic
151
Virion
Only refers to the complete infectious particle within the Virus able to infect the host
152
Reverse transcriptase activities
- RNA dependent DNA polymerase
- 3' Ribonuclease
- DNA dependent DNA polymerase
153
HIV capsid contains
- 2x + ssRNA
- Reverse transcriptase
- tRNA
- Integrase
154
Replication of HIV steps
1) Attachment & Fusion
2) Reverse Transcription (cyt)
3) Integration (nucleus)
4) Transcription, Replication
5) Assembly
6) Budding & Maturation
155
Interferons
- INFa: Leukocytes
- INFB: Fibroblasts
156
What do IFNs induce production of
- Oligo A (oligoadenylate synthase)
- Ribonuclease L
- dsRNA dependent protein kinase (PKR)
157
In-Vivo Gene therapy
- Genes transferred into cells in the patient when cells cant be cultured in-vitro
- e.g. Cystic fibrosis, Spinal muscular atrophy (treated with adeno-associated viral vector)
158
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
159
Non-viral vector types
- Pure DNA construct
- Lipoplexes
- Polyplex
- Human artifical chromosome
- DNA molecular conjugates
160
Therapeutic strategies in Gene therapy
- Gene Augmentation
- Direct/Indirect cell killing
- Targeted inhibition
- Targeted gene mutation correction
161
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
162
CRISPR stands for
Clustered Regularly Interspersed Short Palindromic Repeat
163
CRISPR/Cas9
Used in Bacterial immune system (adaptive) granting resistance against bacteriophages
1st discovered in E.coli
164
CRISPR structure
- Short palindromic repeats (20-50)
- Identical but interspaced
- Unique Spacers between repeats
- Spacers found to be viral/bacteriophage DNA
165
CRISPR associated genes
(cas genes)
- Helicases (unwind)
- Nucleases (cut)
166
CRISPR 1st Viral infection
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
167
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
168
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)
169
Transgenic animal roles
- Disease models
- Transpharmers
- Xenoplanters
- Food sources
- Scientific models
170
Ways of delivery of genetic modification
- Microinjection
- Transfection
- Electroportation
- Retroviral Vectors
171
Knock-in technique
Insertion of a transgene or modified allele producing a gain of function mutation
172
Knock-out technique
Removal / inactivation of a gene to test its function (based on homologous combination where working gene is swapped with another)
- e.g. Neomycin-resistance element (neo-r gene)
173
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 used as a signal for Cre & LoxP
174
Knock-down technique
Partially or temporarily reducing a gene's expression
175
Methods of mRNA degradation in Knock-down technique
- RNA interference (RNAi)
- Hammerhead Ribozyme
176
RNA interference Knock-down (RNAi)
1) dsRNA cut into siRNA by Dicer
2) siRNA binds RISC complex
3) RISC uses siRNA to find complementary mRNA
4) RISC cuts mRNA and its degraded so protein is no longer made
177
Hammerhead Ribozyme Knock-down
1) Design hammerhead ribozyme complementary to mRNA
2) Transfect ribozyme to cytosol
3) Ribozyme binds & cleaves target mRNA
178
TALENs
Transcription activator-like Effector nucleases
DNA binding domain & Nuclease domain
Knicks dsDNA to promote repair
(zinc finger nucleases work the same)
179
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
180
Cloning types
- Reproductive cloning: Create living organism
- Therapeutic cloning: Stem cells for treatment
181
Nuclear receptors
Direct binding to DNA to regulate Gene expression
182
Nuclear receptors main structure
- DNA binding domain (2 Zn fingers: p/d box)
- Hinge domain (NLS)
- Ligand binding domain (AF-2 site act. transcription)
- N-terminal domain
- C-terminal domain
183
Zinc finger
Short DNA helix & 2 short B-pleated sheats
- P-box: Recognizes RE
- D-box: Dimerization
184
Type I Nuclear receptors
Anchored in the Cytoplasm & Bind palindromic repeats
- Steroid receptors
(homodimers)
185
Type II Nuclear receptors
Retained in Nucleus & Bind direct repeats
- Thyroid receptor
- Retinoid Receptor
(homo / heterodimers)
186
Mech. of Type I Nuclear receptors
1) Ligand binds
2) Complex transported by Immunofilin to nucleus
3) SR binds DNA in nucleus
4) Co-activators collected, acetylate histones, DNA unwinds, Transcription
187
Mech. of Type II Nuclear receptors
Nuclear receptor (e.g. Thyroid) always bound to the DNA
- No ligand: Co-repressors bound, Deacetylation
- Ligand: Switch to Co-activators, Acetylation, DNA unwind, transcrip.
188
Xenobiotics
Biologically active chemical substances that are not naturally produced or present in the body (medicines, poisons, pollutants)
189
Xenobiotic Elimination
- Phase-I: Cytochrome-p450 add polar functional groups by oxidizing (-OH, -COOH)
- Phase-II: Conjugated with charged particles by UDP-glucuronosyl transferase (glutathione, sulfate, glycine)
Excretion in Bile / Urine
190
AhR receptor structure
Aryl Hydrocarbon Receptor (cytoplasm)
- Ligand binding domain
- Hs binding site
- DNA binding domain
- Activation of transcription domain
- NLS
Helix-loop-helix structure
191
AhR receptor subunits
- Type I: AhR (xenobiotics)
- Type II: ARNT (HIF-1B)
192
Mech. of AhR receptor
1) Anchored in cytoplasm waiting for ligand
2) Ligand binds
3) Complex transported by XAP-2 to Nucleus
4) Receptor dimerizes with Type II subunit ARNT/HIF-1B
XRE / DRE on DNA
193
What helps Maintain Nuclear receptor strcuture
- Hsp90
- p23
194
Benzo(a)pyrene dangers
Xenobiotic that forms reactive intermediates Diol & Epoxides
- Can intercalate bw DNA bases
- Cis can be fixed by NER
- Trans cant be fixed = mutation
195
Bond bw Ubiq. and Lysine
Isopeptide Bond
