CMB2 Flashcards
1
Q
Cystic fibrosis (caused by a deletion of what )
A
- Deletion of phenylalanine @ residue 508
- Of the cystic fibrosis TM conductance regulator
2
Q
What happens in cystic fibrosis
A
- Causes protein to misfold @ endoplasmic reticulum
3
Q
What goes wrong in cystic fibrosis
A
- The misfiling gets recognized by cellular machinery
- That identifies and processes abhorrent/naughty/misfolded proteins
- This triggers ubiquitinsation
- Which is the process of trafficking to the proteasome and degradation
- This prevents it being placed within the membrane so there is a lack of CFTR
4
Q
what is the name of the misfolded protein
A
- PrPsc
5
Q
How can cystic fibrosis act as infectious proteins
A
- Can interact w normal proteins
- Through this interaction they induce misfolding of the normal protein and polymerisation “bad company corrupts good character”
- Oligomers form fibrils of misfolded protein
- Process relies on energy minimisation concept
- There is interaction of molecules leading to more stable and aggregated structure
- Like putting a neatly folded top on top of lots of messy ones will undergopolymerisation and make fibrils of the messy incorrectly folded ones.
6
Q
what is Alzheimers disease caused by
A
Alzheimers is an instance of protein misfolding
- Proteolytic cleavage
- Of APP (amyloid precursor protein)
7
Q
What are the functions of Amyloid precursor protein (APP)
A
- APP has multiple functions
- APP is involved in G protein signaling
- Cleavage of amyloid precursor protein = @40 residue peptide
- This will anchor the protein in the membrane
- And will cause the brains neuronal cells to die
8
Q
what will Amyloid precusor protein do the the brains neuronal cells
A
- And will cause the brains neuronal cells to die
9
Q
What is beta amyloid
A
- Beta amyloid is a small protein that is released as a result of proteolysis from a larger APP transmembrane protein
- B amyloid forms multimers with a specific structure
- The Beta structure (which is formed rather than the alpha helix) becomes agreagated in plaques
10
Q
what else happens within the Amyloid Hypothesis in Alzheimers
A
- In Alzheimer’s disease the -Amyloid (A) peptide accumulates
- Mis-folding of this protein results in a planar arrangement and polymerisation
- This can form fibrils of mis-folded protein (amyloid fibrils)
- -Amyloid (A) fibres are formed from stacked beta sheets in which the side chains interdigitate
11
Q
what is primary structure
A
covalent bonds that make a polypeptide chain
- The order of amino acid residues
- Joined by peptide bonds
12
Q
what is secondary structure
A
regular FOLDED form
- Often stabilized by H bonds
13
Q
what is tertiary structure
A
overall 3D structure
- Often stabilized by H bonds, plus ionic/vdw/intrachain covalent bonds (disulfide bonds)
- Combining tertiary structures (such as the 7TMD of TSH)
14
Q
what is quarternary structure
A
- organization of polypeptides
- Into assemblies
- Stabilised by non covalent bonds/ disulfide bonds
- Eg there is 4 different polypeptide chains that constitute a functional Hb molecule
15
Q
The nature of the bonds that put them together
A
- The bonds that determine folding are covalent / non covalent bonds
- Weak covalent bonds only have 1/20th of strength of a covalent bond
- But they are much greater in number in comparison
- So non covalents = significant contribution
16
Q
where do disulfide bonds form
A
between the side chains of the cysteine residues
17
Q
what kind of reaction are disulfide bonds made in
A
oxidative reaction
- This forms a very strong covalent bond
- There is cross linkage of the cysteines
- Usually occurs in distant parts of the amino acid sequence
18
Q
Why is the level of structure important to the protein function
A
- The structure of the protein is what determines its potential for molecular interactions
- Chemical composition and constituent AAs also confer structural behavior of polypeptide chain
19
Q
What is the cytoskeleton
A
- The purpose of the cytoskeleton is to help cells do 3 things
- Maintain their shape
- Organize their organelles
- Transport their vesicles
(MOT)
- Transport their vesicles
- Meant to keep and modify the shape of the cell
20
Q
What are the components
A
- Microtubules
- Intermediate filaments
- Actin filaments
21
Q
Microtubules : tubulin tubes
A
- Hollow tubues
- Made of tubulin
- Rigid long and straight
- Organelle positioning
- Intracellular transport
- Each filament is polarized and has a direction + has a dynamic structure
- Assemble and disassemble in response to the needs of the cell
- Tubulin in the cells = roughly 50:50 as free or in filament
22
Q
There is a microtubule organizing centre
A
- MTOC centre
- Specialized protein complexes
- From where the assembly of tubulin units begins
- Centrosome is in the perinuclear region
- The MTOC in most of the cells
- Contains the Y tubulin ring
- This Y tubulin ring will initiate the microtubulues growth
- Heterodimers of alpha and beta tubulin will constitute the microtubules
- It is a polarized growth
- The positive end grows more quickly than the negative
23
Q
Functions of the microtubule
A
- Act as a railway track
- Molecular motors run on them
- There are different motors for different cargoes
- For this the directionaliyu of the filaments is vital (its important which way the train is going!!)
- There is also organization of organelle position
- Hence it provides polarization of cells
24
Q
Microtubule core
A
- Consists of 9 pairs of microtubules
- Around 2 central microtubules
- The motor protein dynein
- Drives the bending of allia and flagella
- The basal body @ the base of the tubule
- Controls the assembly of the axonme
- Microtubules responsible w cilia in respiratory tract
- Sweeping of mucus and debris from the lungs
- There are flagella on the spermatozoa
25
Intermediate filament
- Heterogenous group of filamentous proteins
- Mechanical strength
- Heterogenous group of filamentous proteins
- Mechanical strength
26
Intermediate filament characteristics
- Toughest of all cytoskeletal filaments
- Resistant to detergents (hence why that man is dirty) + high salts
- Intermediate in size (8-12nm) between actin and microtubules
- Form a network throughout the cytoplasm
- Joining up cell to cell junctions (desmosomes)
- Able to withstand mechanical stress when cells get stretched
- The network is also formed in the surrounding nucleus
- Which strengthens the nuclear envelope
27
Polymerization of intermediate filaments
- Each units made up of N terminus head and C terminus tail
- Central elongated rod like domain
- Units form stable simers
- (tough men w a central elongated rod… and are emotionless/ emotionally stable)
- Every 2 dimers make a tetramer
- These tetramers bind to each other
- The tetramers twitst to constitute a rope like filament
- Can get cytoplasmic and nueclar intermediate filaments
Intermediate filament
- Heterogenous group of filamentous proteins
- Mechanical strength
- Rope like w many long strands twisted together
Intermediate filament characteristics
- Toughest of all cytoskeletal filaments
- Resistant to detergents (hence why that man is dirty) + high salts
- Intermediate in size (8-12nm) between actin and microtubules
- Form a network throughout the cytoplasm
- Joining up cell to cell junctions (desmosomes)
- Able to withstand mechanical stress when cells get stretched
- The network is also formed in the surrounding nucleus
- Which strengthens the nuclear envelope
Polymerization of intermediate filaments - Each units made up of N terminus head and C terminus tail
- Central elongated rod like domain
- Units form stable simers
- (tough men w a central elongated rod… and are emotionless/ emotionally stable)
- Every 2 dimers make a tetramer
- These tetramers bind to each other
- The tetramers twitst to constitute a rope like filament
- Can get cytoplasmic and nueclar intermediate filaments
28
CYTOPLASMIC
| where are keratins
in epithelia
29
vimentins
connective tissue muscle cells + neuroglial cells
30
neurofilaments
nuclear lamins + in all nucleated cells
31
nuclear
- Nuclear lamins = in all nucleated cells
32
what is fillagrin
binds the keratin filaments into bundles
33
what do Synamin and plectin do
binds desmins and vimentins –
- links intermediate filaments to the other cytoskeletal compounds
- ie actin and microtubules
- as well as cell-cell contact structures
- desmosomes
34
what do plakins do
-keep contact between the desposomes
| planking between desmosomes
35
where are the intermediate ropy strong filaments in the nucleus
- Present in all nucleated eukaryotic cells
36
Functions of the intermediate ropy strong filaments in the nucleus
- Form a mesh rather than a rope like structure
- They line the inner face of the nuclear envelope
- This lining of the nuclear envelope will
- 1. Strengthen it
- 2. Provide attachment sites for chromatin
- Able to disassemble and reform as the nuclear envelope disintegrates
- V different from the stable cytoplasmic fragments
- Process is controlled by post translational modification
- Mainly phosphorylation and dephosphorylation (taking on and off phosphate groups)
37
The functions of intermediate ropy strong filaments in the cytoplasm
- Tensile strength
- This enables the cells to withstand mechanical stress to stress
- Structural support by
1. Creating a deformable 3D structural framework
2. Reinforcing cell shape and fixing organelles localization
38
characteristics of Actin filaments /
| microfilaments
- Cell shape
- Organelle shape
- Cell migration
- Made of actin
- Flexible and is organized into 2D networks
39
characteristics of micr
- Monomers are abundant
- Not covalently linked
- Has accessory proteins
- Which regulate the size and rate of filament formation
- Polymerirastion and depolymerisation
40
functions of actin filaments
Cytokinesis – involvement of an actin myosin ring
Cell migration – a multistep process
- Cell pushes out protrusions
- At its front
- Lamellipodia and filiopodia
- Actin polymerization
- Protrusions can adhere to the surface
- Integrins can link the actin filaments to the extracellular matrix that surrounds the cell
- Cell contractions and relaxations
- Of the rear part of the cell
- There is interaction between actin filaments and myosin
41
what is G actin like
- The ones that are twisted are G actin
| - G actin makes up the filamentous form which is F actin
42
what is F actin like
- F actin = skinniest type (ariana grande) – at 7nm each it’s the skinniest
- This presents as structurally polar
- Its associated w a large number of actin landing proteins (ABP)
- Variety of orgnaisations and functions
43
how many isoforms does actin have
- Variety of orgnaisations and functions
44
what are the characterstics of these isoforms of actin
- Each isoform has a different isoelectric point
- ALPHA actin = found in many of the muscle cells (alpha males = lots of muscle)
- Beta and gamma actin = found in non muscle cells
45
Actin polymerisation
- Actin polymerization can occur
- Actin filaments (F actin)
- Can get longer and longer by adding actin momers
- The filament length = determined by the concentration of G actin
- And the presence of actin binding proteins
46
What are Actin binding proteins (allow the growth of the F actins and will keep them in parallel bundles – keeps them in check)
- Proteins that bind to monomoers
| - Keep the F actin (filamentous actin) in parallel bundles
47
Control of G actin monomers
- Controlled by 2 actin binding proteins (ABPs)
PROFILIN (MC – high profile and has lots of pics taken of her profile )
- Profiling facilitates Actin polymerization
THYMOSIN beta 4
- Prevents the addition of actin monomers to the F actin (the thinnest type)
- (being reminded of sin stopped her growth)
48
Actin binding proteins
- F actin (skinny type) severing proteins
- These will BREAK F actin into smaller filaments
- Motor proteins (myosin)
- The transport of vesicles and or organelles
- Through the actin filaments
49
Functions of actin filaments - skeletal muscle
- Arranged in paracrytalline array and integrated w different actin binding proteins
- Interaction w myosin motors
- Allows for muscle contraction (alpha actin)
50
Functions of actin filaments - non muscle cells
- Cell cortex
- From a tin sheath
- Beneath the plasma membrane
- Associated with myosin to form a purse string rig
- This results in a purse string – resulting the cleavage of mitotic cells
51
what is PCR
- Enzyme based method
- Specifically amplfys DNA segments
- By using thermal DNA polymerase
- In a cyclical process
52
How does PCR work
- DNA polymerase syntheseis new DNA strands
- By making a complementary cope of the opposing template strand
- Dna polymerase recognizes a specific structure that constists partially of double stranded DNA
- This double stranded DNA forms an invitation complex with it
- The reaction extends a partially double stranded molecule , from the 3’ end of 1 strand
- Each cycle leads to the doubling of the amount of product
- Have to have SNP detection
- This depends on the differnces of the melting temperature (Tm)
- That is conferred on these short DNA sequences of DNA by the nucleotide
- Applications
- 1. Antibiotic resistance testing
- 2. Identification of genetic markers (drug sensitivity and catabolism)
- CYP2C9 + VKORC1 variants confer warfarin sensitivity
- 3. Markers of disease (cancner)
- 4. Treatment response
- SNPs can be detected using HRM (high resolution melting)
- Can get the Tm of the amplified produce
- Can be used to determine which sequence is present
53
What is PCR based on the principles of
Based on the process of
1. Denaturing
2. Annealing
3. Native state @ optimal extension temperature for enzyme activity, remaking the double helix
54
What is the diagnostic purpose of PCR in TB
- PCR used for quantification, identification, confirmation
- Of a specific DNA sequence
1. Presence absence calling TB-
Detection in sputum
- Determining treatment
- Response and efficacy
55
What is the diagnostic purpose of PCR in swine flue
- Differentiating between closesly related organism
| - They are BOTH H1N1 subtypes
56
What is the diagnostic purpose of PCR in HIV viral load
- Determines when treatments might be commenced
57
what is the diagnostic purpose of PCR in Forensics and law enforcement
- Checking parentage / kinship
- Identifying military casualties / missing persons
- Matching 2 sources in crime scenes
- Authentation of biological materials eg cell lines and purity of food
58
Explain Forensic identification in more detail
- Uses repetitive sequences
- 2-5 bases long and repeated many times
- At specific locations of the genome
- STRs are highly polymorphic
- Number of repeats varies w individuals
- A molecular barcode is produced
- Pattern of uniquely stored products
59
Discuss importance of PCR in modern biomedical sciences
- Has many other applications
- Such as
- 1. Next gen sequening
2. isolating individual DNA segments (prior to cloning/ sequencing)
3. manipulation/ modification of DNA
- changing it at the ends
To make it contain restriction sites or cloning vectors
- One of most commonly used and important tools used in recombinant DNA technology
- Eg in developing recombinant DNA vaccines + pharmaceuticals
60
What is the Lac Operon that controls gene regulation
- The lac operon is a transcription factor protein that controls the transcription of bacteria such as Ecoli
- It allows the breakdown of lactose
61
what is Lac Operon made up of
- The system is composed of a
- Promotor region
- Operator region
- Repressor region
- Gene
62
What happens to Lac operon when it is turned on
- When switched “on” the repressor region is binded to the operator region and will block the movement of RNA polymerase
- The presence of lactose causes the repressor to be “switched on”
- It will change shape and will move away from the the operator
- Thus freeing the RNA polymerase to move downstream and produce RNA from the DNA strand.
63
What types are RNA found in a mammalian cell
Eukaryotic RNA
- mRNA
- tRNA
- miRNA
- rRNA
- small RNAs
- non coding RNAs
64
Eukaryotic RNA
- mRNA
- tRNA
- miRNA
- rRNA
- small RNAs
- non coding RNAs
- mRNA = mature RNA that is produced in transcription and is used to produce the protein in translation
- tRNA= transfer RNA that is used to transfer amino acids to the single strands of RNA in transcription
- miRNA = act to control the post transcriptional regulation of almost a 1/3 of human genes. Can regulate several target genes.
- microRNA derived by processing from larger processors
- they search for complementary target mRNA
65
what is a point mutation
- When a single base is changed
| - Causing the amino acid that is coded for to be altered
66
What are the components
- Group of genetic disease
| - Caused by the insufficient expression of B gobin
67
Define what gene expression is
- Differential gene expression means that a genome has to be different interpreted in different cells @ different times
- Genome contains 50k genes and only about 10k are expressed bc all human cells contain the same blueprint
68
Outline the synthesis of a protein
- Cell differentiation decides whether a cell will become a blood vessel / brain / muscle cell
69
ow the expression of a gene can lead to protein synthesis
- Drosophilia homeotic mutant
- Bithoradx – the expression of a pair of wings from the abdomen
- Antennapedia – gene needed to form legs in flies
- If they are not expressed in the correct place then there can be legs sticking out of the head
70
How can wrong gene expression
- Group of genetic diseases
- Caused by expression of Beta globin being insufficient
- In most cases beta globin proteins are structurally normal unlike in SCD
- There are multiple independently arising forms of disease
- The mutation causes Beta thalassaemia map to multiple sites in the B globin gene
71
what are the defintions of isoenzymes
- Isozymes - Multiple forms of enzymes)
- = enzymes that differ in amino acid sequence but catalyze the same chemical reaction.
- These enzymes usually display different kinetic parameters (e.g. different KM values), or different regulatory properties.
72
How are isoenzymes used as diagnostic tools in DNA technology
- Restriction endonucleases
- Can 1. Recognize specific sequences
- 2. Cut this specific sequence
- Restriction can limit the transfer of nucleic acids from infecting the phages into bacteria
- There are many different enzymes from different bacteria
- Restriction enzymes can catalyse the hydrolysis of phosphodiester bonds
- DNA ligase can repair nicks in phosphodiester bonds
- DNA polyermase
- Synthesesis of DNA downstream (5’ to 3’)
- Copies of DNA are made
- DNA polymerase is used in PCR reactions to make a lot of DNA from a single strand
- DNA polymerase can be used for
- 1. PCR amplification 2. Generating probes 3. Blunt ending DNA overhangs
- Can add complementary labelled fragments
73
More useful enzymes in DNA technology
| Phosphatase
- Phosphotases hydrolyse phosphates off the substrate
- Calf intestinal alkaline phosphatase
- Remove phosphates from the 5’ end
- Used to prevent plasmids that have been cut from resealing back up again
- If they were to close up again then the insert would not be able to be taken – a gap is needed inside
- ++ of phosphatases is that the fragment has more chances to get into the plasmid
- Want the fragment to close WITH the cut inside
74
More useful enzymes in DNA technology
| Polynucleotide kinase
- Kinase takes phosphates from ATP to the substrate
- Polynucleotide kinase adds phosphates to the 5’ hydroxyl group of DNA or RNA
- Can be used to phosphorylate chemically synthesized DNA
- This is so that the phosphorylated DNA can be ligated to another fragment
- Polynucleotide kinase can be used to sensitively lable DNA
- So that it can be traced using 1. Radioactively labelled ATP
- 2. Fluorescently labelled ATP
75
More useful enzymes in DNA technology Reverse transcriptase
- RNA dependent DNA polymerase
- Isolated from RNA that contains retroviruses
- Reverse transcriptase is the only enzyme that can copy DNA from an RNA strand
- DNA molecule is synthesissed
- That is a complementary to an mRNA strand
- Using Dntps (deoxynucelotide triphosphates, Dgtp, dctp, datp, dttp)
76
Discuss the modularity of motifs in transcription factors - What is a transcription factor
- Transcription factors
- Are proteins that bind DNA and regulate transcription
- They can also switch genes on/off and downregulate genes
- Many transcription fctors
77
How do motifs come into transcription factors
Many of them have the chemical characteristic of being BASIC.
They are basic because of the acid nature of DNA (ie a proton doner) and is negatively charged therefore interaction of DNA binding motifs require attractive forces such as electrostatic forces to operate in binding and recognition.
- The amino acids on the face of the recognition helix opposing the DNA tend to be rich in basic amino acids like Arginine and Lysine, this is because they have positively charged amine groups which make specific contacts with the DNA which is of course negatively charged.
- Many transcription factors
- Contain a small number of conserved motifs that combine
- These motifs combine to form domains
- These domains interact with the DNA
78
Motifs
- The motifs get conserved across all the phyla
- And form DNA binding domains that allow regulatory function of the respective proteins
- Proteins are formed from homo and heterodimers
- Can identify motif types and can work out if there is a type of protein that is a transcription factor or a regulator
- Actual binding to DNA is important to the recognition of the specific gene
- Transcription factors can form proteins either from 2 similar or 2 idfferent polypeptide chains
- Which forms either homodimers or heterodimers
- They can come together in diff combinations that recognize different types of promoter sequences
79
Alpha helices = important to DNA binding how?
- Can fit in the MAJOR groove of DNA
- This groove is larger than the others – so can fit the alpha helix into it
- This is the one that makes contact w the amino acid
- There are various chains that stick out of the alpha helix
- That makes specifc contact w the bases (other parts make non specific contacts)
- There are other parts of the protein that are critical to 1. Stablisiation and 2. Contributions to reactions
- Amino acid transcription factors can give specificity of binding to genome & particular gene
80
describe Helix loop helix motif
- Binds the DNA in dimeric form
- Exists as hetero and homodimers
- Heterodimers = made up of different monomers
- Homodimers = identical monomers
- The central portion is formed from the helices that overalp
- This dorms a structure that enables dimerization
- Terminal part of the lower opposing helice contains basic amino acids
- These intercat with the major DNA groove and gives rise to the b/HLH domain
= polypeptides come together and straddle the amino acids
81
Leucine zipper motif
- 2 continuous alpha helices
- Dimeric protein (2 sides of a zip)
- Formed from 2 polypeptide chains
- At the top stalk the dimers zip together
- This forms a short coiled coil
82
how is the coil held together in the leucine zipper motif
held together by hydrophobic interactions, down opposing sides of the helix
83
What occurs in the helix of the b/HLH domain
- Basic amino acids will dominate the lower part of the helix
- And can interact w/ DNA major groove
- Heterodimerisation expands the regulatory potential of the leucine zippers
- Formed from 2 polypeptide chains
- Palindromic sequenes are NOT recognised
84
what is likely to have a helix turn helix domain
a repressor
85
does helix turn helix have the same appearance as the helix loop helix or leucine zipper
no
| - Structural appearance = different
86
what is the structure of the helix turn helix domain
- Arrangement of 2 SHORT alpha helices
- Orientated @ right angles
- Connected by a turtn
- Motif is found in both prokaryotic and eukaryotes DNA biding proteins eg CRO repressor and homeobox proteins
87
what is the homodimer of helix turn helix domain
- CRO protein = homodimer
| - CRO recognizes the palindromic sequence and by binding DNA will repress transcription
88
what happens once the recognition helix interacts with the nucelotide sequence
- Once the recognition helix interacts w the nucleotide seuqnce itself other contacts are made with the phosphate backbone
- Then locates within the major groove
- Being at right angles is critical for presentation
89
give 4 examples of where the zinc finger motif is found
- Glucocorticoid
- Mineralcorticoid
- Oestrogen
- Progesterone
- Vitamin D receptors
90
4 marker on the structure of the zinc finger motif
- An alpha helix AND a beta pleated sheet
- Held by non covalent interactions with ZINC – the fingers are what hold things together when you pick them up
- Dimer w 2 separate motifs on separate polypeptide chains
- Each polypeptide chain contains 2 zinc atoms
- These zinc atoms stabilize the recognition helix and loop structure
- The alpha helix of each motif = interact w major groove of DNA
- DNA can recognize specific DNA seq
- Arrangements of polypeptide chain around the molecule
- The ones at the centre = will stabilize the overall structure
- The ones at the end will stabilize the struture where the recognition helix
91
what is electrophoresis
- technique for separating charged molecules in an electric field. This is achieved by loading a mixture of molecules typically proteins or nucleic acids on into a matrix and applying an electric potential.
92
whats the deal with amino acids properties
- Amino acids = building blocks for proteins
- There are 2 ionisable groups
- Amino group = N terminus
- Carboxyl group = C terminus
Therefore can act as both acids and bases in solution @ typical pH
- Results in formation of zwitterions
- Zwitterion = molecule w BOTH ionized amino and carboxyl ends
- The pH= electrically neutral. This is called the isoelectric point.
93
what is the isoelectric point
point that the molecule is electrically netural – water is neutral”
94
how is the protein backbone charged
- Protein backbone is not charged
- Proteins can have net negative or net positive charge
- The protein charge depends on buffer pH
- R group determines whether amino acid is neutral acidic or basic
95
give examples of the basic amino acids
- Histidine
- Arginine
- Lyseine
96
acidic amino acids
ACIDIC AMINO ACIDS
- aspartic acid
- glutamic acid
97
what happens when the pH is less than the pI
- will be acidic solution because its less than neutral value
- therefore a net positive charge
98
what happens when ph is more than pI
- when pH more than Pi (isoelectiv point @ which molecule is neutral) – will be a basic solution and theres a net negative charge
99
What actually happens in electrophoresis
- migration
- of charged particles (macromolecules)
- in electric field
- migration based on size shape and size + current and resistance
- process = useful for separating macromolecules
- eg proteins and nucleic acids
- macromolecules have lots of different subunits
100
what is the separation of electrophoresis based on
- separation based on size shape and charge
| - current and resistance (fat ones may have greater resistance than skinnier ones)
101
what is electrophoresis based on
- process = useful for separating macromolecules
| - eg proteins and nucleic acids
102
why is electrophoresis good @ separating macromolecules
- because macromolecules have lots of different subunits
103
what is included in horizontal gel electrophoresis
- horizontal – usually for agarose gel
| Agarose gel LARGE ROSE = IT SEPARATES LARGE PROTEINS – Planted horizontally
104
what does agarose gel separate + what are its characteristics
- large protein separation
- polysaccharide extract from seawed
- dissolve powdered agarose in bugger
- heat and pour into casting tray
- polymerization occurs when cooled
- has relatively large pores – size determined by concentration of agarose.
105
what is included in vertical electrophoresis
- vertical – polyacrylamide gel
- polyacrylamide gel polyacrylamide = a long word so it is verticlal and really tall)
- acrylamide
- polymerises into long chains
- smaller pores than agarose
- pore size determined by conc of polyacylamide
requirements
106
Steps of gel electrophoresis
1. Protein gets electrophoresied within the matrix / gels in a series of pores
2. Gel = thin slabs within the well
3. Proteins immersed within a buffer – buffer provides ions to carry current + maintain constant pH
4. pH of solution + how the R groups are have an important effect on the migration of proteins
107
What are the types of buffer systems and what do the buffers do
- buffers supply current carrying ions in electrophoretic cells
- this maintains the desired pH
- a buffer lets the heat get dissipated across it
- either continuous or discontinuous buffers
- continuous = same buffer that is within the gel (it continues in the same medium as the gel)
- Discontinuous = different buffers. Large resolution
108
what is SDS page
- sodium
- dodecyl
- sulfate
- polyacrylamide
- gel electrophoresis
- The migration of the protein is not determined by the intrinsic electric charge
- Protein migration is determined by weight:
109
what are the downsides of SDS page
- cannot be used on small polypeptides + peptides
- with a molecular weight less than 10kDa
- can use continuous / discontinuous sequence
110
characteristics of SDS page
- = most common
- It’s a strong & anionic detergent
- It solubilizes, dissociates, and denatures
- Most proteins
- To single polypeptide chains
- Disrupts H bonding
- And blocks hydrophobic interactions
- Binds @ ratio : 1.4g SDS to 1g Protein
- SDS page confers a net negative charge to the polypeptide
- This is in proportion to length
- SDS page includes disulfide bond cleaving agents
- The migration of the protein is not determined by the intrinsic electric charge
- Protein migration is determined by weight:
111
how does movement of macromolecules happen in SDS page
- They move in an electric field
- Negatively charged molecule eg DNA towards ANODE (positive bit)
- Positive charge cathode - opposites will attract
112
Detection methods of SDS page
- Staining of proteins
- Fluorescent staining
- Silver staining
- Radioactive methods (radiolabelling / autoradiography/ fillorography)
113
what is native / non denaturing gel electrophoresis
- Runs without SDS (naked)
- Proteins don’t get denatured
- Separation = based on charge to size ration and conformation
- Charge changes w charge of buffer pH
114
This native gel electrophoresis = advantages
- Separates proteins w the same Mw
- Protein recovered in the native state and not single strands
- Can study binding events (if it’s a protein – protein or protein - ligand)
115
give examples of native gels
- Agarose (horizontal) and polyacrylamide (vertical) are native gels
- Polyacrylamide gels = do have uniform pore size
116
what weight of proteins is polyacrylamide used for
5-2000kDa
117
Clinical applications of electrophoresis
Serum protein electrophoresis
| Haemoglobin electrophoresis
118
Serum protein electrophoresis
blood is made up of plasma & blood cells
- Plasma = proteins / salts / glucose / hormones / clot factors
- Meaures certain proteins in blood
- Uses electrical field to separate proteins into similar shapes / sizes + charges
- Helps w disease identification
- Blood plasma contains albumin and globulin
119
Haemoglobin electrophoresis
- Buffer system usually alkaline
| - Majority of proteins = negatively charged
120
give a summary of denaturating cell electrophoresis of proteins
- Strong anionic detergent
- Dissociates and denatures proteins to make polypeptides
- Proteins will infold + produce similar rod like shapes
- SDS complexes w protein and masks protein charge
- Separation based on molecular size
- Proteins resolved can get detected by different stains
121
What is chromatography
- Separation method in which components are partitioned between a moving and stationary phase
- Uses charge dist, molecular size, solubuily, binding properties (CMSb)
- The isolation of protein
- From 1000s
- To be able to study its properties
- Can be used on large quantities first and then use methods only for small amounts
122
Chromatography - SICE separation techniques can be used
Salting in and out
Isoelectic focussing
Column chormatography
Electrophoresis
- Sample = dissolved through mobile phases
- The its forced to get through the stationary
- Proteins are the nseparated and their properties observed
123
What is the purification protocol for proteins
- Determined by getting 2+ properties and combining them
124
what is the method of ion exchange chromatography
- Separation of larger amounts of material
- Usually have a charge group attached to the stationry phase
- This charge group is either acidic or basic
125
what is the method of Cation exchangers in chromatography
- The acidic group of resin are called cation exchangers
- Cation exchanger – separate positive charge proteins
- Anion exchanger – sepeartes negative charge proteins
126
How does IEX chromatography work
- More negatively charged samples = bound to column
- Go through more quickly
- Positively charged ions bound to column
- Can separate based on how tighly they are bound to the column
- Proteins = bind to ion exchanger that has different affinities
- All about affinities
127
explain the flow of proteins of different sizes in IEX chromatography
- As column gets washed w the buffer-
- proteins w lower affinities for the ion exchange resin will move through column faster (less stuck to the sides)!!!
- + charge ions bind to column
- Protein bind to ion exchange
- Proteins can get eluted by changing the elution bugger to one w higher salt [ ]
- Those that are not eluded have to be eluded using high salt buffers
128
Gel filtration chromatography
- Separation based on size /shape
- Large molecules elute first bc they cant fit into the matrix
- Known as a reverse sieve
- Smaller particles enter matrix + stay there fore longer
- Matrix has really small pores
- Usually is used @ the end of the purification process
- To separate a protein of interest and get rid of denatured proteins
- And keep the correctly folded one
129
What is the elution volume
- Volume of a solvent needed to elute a given solute from column
- After it has first contacted the gel
- (eg elution volume is how much you would have to hose an intruder until they left your house). Larger molecules = smaller - Volume of a solvent needed to elute a given solute from column
- After it has first contacted the gel
- (eg elution volume is how much you would have to hose an intruder until they left your house). Larger molecules = smaller
130
Affinity chromatography basis
- Many proteins can bind specific molecules non covalently
| - Based on the reversible interactions between the target protein and specific ligand
131
what is Affinity chromatography used for
- Can be used to separate / isolate
| Proteins, Antibodies, Antigens, Hormones
132
What are the requirements for affinity chromatography
- Beads matrix eg agarose
- Ligand molecule to specifically bind to the protein that you are interested in
- It will join to the protein and make it more visible
- Lizzie= protein of interest and jumper is the ligand that binds and makes her more noticible.
- The ligand can make binding to the proteins that we actually want possible not binding undesired proteins
133
process of affinity chromatography
- Solution that contains the substance has to be isolated
- This then has to be isolated and then washed
- To elute and get rid of any unbound substances
134
What is isoelectric focusing
- Pouring in of proteins
- They’ll stop moving when they reach their isoelectric points
- Theres descending isoelectric points on column
- Sample put into SDS
135
High performance liquid chromatography
- High performance forced w a lot of pressure put on it
- Forced @ a pressure of 400atm through the column
- Allowing use of smaller particle size for column material
- Causing a rise in surface area
- This allows better separation of the componenets
- Can use a really small amount of the protein sample but STILL be able to distinguish the proteins
136
advantages of High performance liquid chromatography
- Good : fast + high res, sensitive, reproducible, accurate, and automatic
137
disadvantages of High performance liquid chromatography
- Bad : expensive, complex, coelution (2 or more chemical compounds going at the same time and getting mixed together – defeats the point)
138
Normal phase chromatography
- The stationary phase = polar
- Mobile phase = less polar (increasing in polarity)
- Decreasing retention
- Solid phase = silica which is available @ low costs (like the snow = silica beads- snow is solid)
- Silica has an OH group on its surface
- (snoooh)
- Increasing polarity and decreasing retention
139
Reverse phase chromatography
- Hydrophobicity
- Non polar functional group gets bonded to silica “bonded phase chromatography”
- Hydrophobic proteins bind to the stationary phase
- Hydrophilic proteins do NOT bind and wash out
- Non polar bear with silica on it
- Hydrophobic proteins
140
what does trancription make
- Transcription is making mRNA transcript from DNA
141
what does translation make
- Translation is making a protein from the mRNA
142
What do chromosomes do in this case of protein synthesis
- They carry genes from one cell generation to the next one
143
Homologous chromosomes
- Carry genes
- Centrosomes allow attachment to the mitotic spindle
- 2 chromosomes- one maternal and one paternal
- Telomere caps the chromosome and protects the chromsomes
144
What is in the intergenic region (between the genes)
P seudogenes
E ndogenous retroviruses
R epetitive DNA
T ransposons
145
Unit of heredity
- Unit of hereditary = the blueprint to make an organism
| - Instructions for organisms phenotype how they outwardly present
146
DNA segment + what direction is 5' and 3'
- Containing instructions
- To make a certain product
- Which isn’t necessarily a protein can function in RNA
- Genes = are not just the parts that code for proteins
- Starts on the 5’UTR side as part of the first exon – gene is a DNA segment that contains instructions for making certain DNA product
- Including the regulatory elements
- Upstream is towards the 5’ end
- Downstream is towards the 3’ end
147
Gene types and sizes
- Histone genes are small
- Dystrophin genes are large
- Genes differ in # of exons and introns
- Genes can cluster into diff families
148
what is the action of RNA polymerase
- RNA polymerase transcribes
- DNA RNA
- A small portion of the DNA is unwinded, and there is 5’ to 3’ synthesis
- Due to Watson crick base pairing rules
- Production of phosphodiester bonds = catalyzed by RNA polymerase in the 5’ direction
- The m RNA produced = single stranded and goes in the downstream direction to the 3’ end
149
RNA polymerase 1 makes
rRNA
150
RNA polymerase 2
miRNA
Protein coding RNA
non coding RNA
151
RNA polymerase 3
tRNA
5s RNA
Other small RNAs
152
RNA synthesis
- Quick
- 1500 nucleotides in 50secs
- Dozens of polymerases work on same gene
- Many transcripts come from a gene @ same time
- Cell might need to quickly produce more proteins
153
what are transcription factors
- Proteins
- That are needed to initiate transcription in eukaryotes
- TFs assemble on the promoter
- And transcription factors position the RNA polymerase 2
- RNA polymerase pulls apart the DNA helix and will expose the template strand
- Transcription factors will position the TATA box
- The tata box is recognized by the TATA binding proteins
- TFIID is a TATA binding protein that distorts the DNA
- RNA is unable to attach itself @ DNA strand bc it’s not effective at binding to the DNA
- + TATA box needed to recruit the transcription factors
154
Basal promoters and general transcription factors
- TATA box recognized by TBP (a subunit of TFIID)
- TFIID binds via the TBP subunit (dog bites the TFIID)
- This distorts the DNA
- TFIIA binds
- Other factors get recruited including TFIIH following recruitment of polymerase TFIIH
- TFIIH phosphorylates RNA polymerase
- TFIIH phosphorylates RNA polymerase
- releases RNA polymerase from the inhibitory complex to produce RNA
- TATA box forms a basal promoter
155
TATA BOX
- 30BP in
- 30bp from the 5’ start site
- One binding shape of DNA will change
- So other promoters can come in and bind, causing other factors to also bind
- = transcription initiated concepts
156
The gene specific regulation of transcription
- Additional upstream sequences are needed for gene specific regulation of transcription
- Polymerase doesn’t bind to genes at the same time and has to be regulated, some genes are turned off/on
- UTR= transcribed and end up in the primary transcript but are NOT translated
- 5’UTR= regulation of translation
- 3’UTR= m RNA stability / regulation and miRNA biding
- The primary transcript is a strand of mRNA that is complementary to the antisense DNA strand
157
describe the primary mRNA transcript
- The transcript gets processed before translation that occur
The primary m RNA transcript that is made during transcription isn’t the final product that is translated into a protein
158
- RNA processing steps
- Capping
- Polyadenylation
- Splicing (cutting out the bits that aren’t needed)
159
what happens in the capping process
- Modification is at the 5’ end
- Guanine nucleotide has a 5’ 5’ triphosphate bridge and is methylate @ the 7 position
- Capping the enzyme complez
- There is co transcriptional modification
160
Polyadenylation
- There is a cleavage signal
- and then there is cleavage by a specific endonuclease
- and addition of a tail by a poly(A)polymerase
161
RNA processing
- Splicing = process of taking out the introns and joining exons
162
Introns
- Non coding sequences
- Introns = so internal that they cant be seen so they aren’t coding
- Histones don’t usually contain introns
163
Splicing
Goal : to end up with 2 exons as a final product
- Happens from the 5’Splice site to 3’splice site
- The 5’SS curls around to the branch point
- Then there is cleavage of the 3’ SS
- 5’ end has a signal at the 3’ end
```
Step 1
- 5’ SS
- Attachement occurs
- Wraps around and joins
Step 2
- Cutting the AG
- Exons 1 and 2 join together
- Degradation of the lariat intron
- Lariat wraps around so that the ends of exon 1 and 2 are in v close proximity
```
164
Alternative splicing
- Exon skipping about 95% of genes
| - Allows splicing to alternative introns and exons
165
The m RNA molecules are exported from the nucleus - involving what
- CBS = the cap binding process
- TREX = the transcription coupled export complex
- EJC= the exons junction complex
3 complexes are involved in the export of mature RNA from the nucleus
166
in the control of genetic expression how many genes does the genome have vs how many are expressed
- Contains 50k genes
- Only 10k expressed
- We all have differential expression
167
- Contains 50k genes
- Only 10k expressed
- We all have differential expression
- time and space
168
how does time influence regulation of gene expression
TIME
- Different development in embryos and adults
- Genes expressed in emboys aren’t the same as in adults
- This is in response to hormones/ infections and different signals
169
how does space influence regulation of gene expression
SPACE
- Diff tissues and cell types express diff genes
- Eg the brain and muscle cells
170
what do proteins made in differential gene expression go on to do
- This protein then goes on to affect things like metabolism/metastasis. Congential disorders and cancer etc
171
Why is control of expression important
- If it wasn’t there then the correct enzyme will not be made and it’s the enzymes that carry out tasks
- Cells cant metastatise if they do wrong thing @ wrong time cell must grow at a rate that is tightly controlled
172
what does the Drosophilia homeotic mutant cause
Bi thorax
- This is the expression of a pair of wings from the abdomen
Antennaepaedia
- is the gene that is needed to form fly lefs
- If the gene isn’t expressed in the right place then legs can stick out of head
173
what do totipotent cells give rise to
any cell
- All of the cells that form are pluripotent
- And can produce all tissues that are required
174
why can pluripotent cells can differentiate into every different tissue
- Due to specialization
175
what is the goal of differentiation
- Isolate adult stem cells
- From developmental compartments
- To drive these cells to develop either in vivo / in vitro
- In a controlled way so that we can regenerate tissues
176
B thalassaemias - what are they
- Group of genetic diseases
- Bc of not enough expression of beta globin
- Beta globin protein = actually normal in structure in most cases
- Mutation causes the beta thalassaemia map to multiple sites @ Beta globin gene
- There is a single base change
- An AG
- In the coding region
177
- There is a single base change in beta thalassaemias - what is it
- An AG
| - In the coding region
178
Give some more examples of translational control
- Early embryogenesis
- Environmental stress
- Exposure to heat shock and pathogens
- Can cause global changes in translation
179
which codon sets the frame of mRNA what what is this codon always?
- The 1st codon sets the frame
| - This first codon is ALWAYS AUG
180
what precedes the AUG framing sequence
kozack sequence
181
how does the 5' upstream area control transcription
plays a role in how efficiently the ribsome initiates translation
- But it doesn’t say whether transcription will be initiated or not
182
how efficient is globin translated
very efficiently
183
is ferritin efficiently translated
- Ferritin = v Inefficiently translated (protein that stores iron and releases it) the intracellular iron levels are therefore translationally controlled - Ferritin is inefficiently translated
- So it will bind iron and stays in the cytoplasm as a store for excess
184
when is ferritin needed
- Ferritin = only needed in times of iron excess Ferritin mrna stays in @ times of excess and comes out in times of starvation
185
Role of the 3’ UTR downstream
- 3’ UTR play role in mRNA stability
- 3’UTR= confers v different stabilities on mRNAs
- Globin 3’ UTRs confer stability
- Immune stress hormones = very unstable mRNAs
186
Small non coding RNAs
| MiRNA role
- miRNA act to control the post transcriptional regulation of up to 1/3 of human genes
- any given miRNA can regulate many target genes
187
how do miRNA come about
- miRNA = come about through processing from a larger processer
188
Mechanism of action of miRNA
- search for complementary target mRNA
- when they find an extensive match its extensively degraded
- when it’s a less extensive match
- transduction is reduced
- mRNA sequestered and is eventually degraded
189
what is the transcriptome
- transcriptome = segment
| - that actually gets transcribed
190
how much of transcriptome is transcribed
- only a fraction of the trancriptome is transcribed at any one time
191
are all genes transcribed to the same level
- genes can get transcribed to different levels can have an abundant transcript, a rare one, or no transcript.
- Abundant transcripts if is “ON” in every cell then this is a housekeeping gene eg glycolytic enzymes .
192
What does transcription need to happen, in relation to transcriptional circuits
- Recognition sequences in DNA
- That lie outside the transcribed region
- Sequence that is immediately 5’ and upstream to the region that’s getting transcribed is the promoter
- the promoters recruit RNA polymerase
- transcription factors flock around the promoter and position this RNA polymerase
- RNA polymerase can only move in one direction
- The promoter region will recruit at the 5’ end and unwind it from this end
- To be able to copy one of the strands
193
Enhancers - how do they work
- promoter is more like to recruit when enhanced / wearing makeup
- Not immediately adjacent to the start of transcription
- Enhance RNA polymerases recruitment to the promoter
- The enhancers can reside either 5 or 3’ to transcription unit
- Enhancers can be locked within an intron (internal bit that isn’t transcribed)
194
what are the characteristics of enhancers
- Not immediately adjacent to the start of transcription
- Enhance RNA polymerases recruitment to the promoter
- The enhancers can reside either 5 or 3’ to transcription unit
- Enhancers can be locked within an intron (internal bit that isn’t transcribed)
195
RNA polymerase has to get recruited to the promoters - why is this and what happens
- Neither prokaryotic / eukaryotic RNA polymerases can make stable contact w the DNA
- Prokaryotic and eukaryotic RNAs will slide along the duplex without being able to recognize the promoters
- The actual recognition is mediated by initiation factors
196
so what does RNA polymerase actually do
- Prokaryotic and eukaryotic RNAs will slide along the duplex without being able to recognize the promoters - needs RNA polymerase to be able to recongise the promoters
197
what initiation factors are used in prokaryootes
- Prokaryotes = the sigma initiation factors
198
what initiation factors are used in- Eukaryotics
the TFII basal transcription machinery “with.. machinery”
199
what happens When the RNA has been stably recruited to made DNA
- RNA polymerase converts from a closed to an open complex.
- Process starts bc these recognition factors
- (sigma for prokaryotes and TFII basal transcription machinery for eukaryotes)
- are used to make RNA and DNA recognize each other
200
within prokaryotic promoters what is - a consensal sequence
```
similar in lots of different places
protein binding site
- may be a short nucleotide sequence
- found on the genome
- for the sigma transcription factor
- to identify the promoter
- the consensual sequences are both needed
```
- here the sigma factor recognizes the -35 and the -10 motif that are common to prokaryotic promoter
- therefore the RNA can make more stable contact w the DNA
201
The TATA box
- consensal sequence
what is it needed for
- needed to recruit the transcription factors
- so that they can carry it
- this is a consensal sequence
- TBP recognizes the TATA box
- It is the TATA binding factor protein
- Eukaryotic RNA polymerase 2 needs general transcription factors
202
Regulatory transcription factors - what do they do
- The ability of sigma factors (prokaryotes) and TF2 to recruit the RNA polymerases to promoters are generic happen @ every promoter
- Able to vary level of transcription from a promoter
- But this doesn’t account for this ability
- Regulatory changes are mediated by different classes of transcription factors (regulatory transcription factors)
- In pro karyotes and eukaryotes
- There is a function to alter to level of RNA recruitment
- And its ability to initiate transcription
- In eukaryotes they can influence local chromatin structure
203
Transcriptional switches
| give a prokaryotic and eukaryotic example
- Prokaryotic switch = lac operon
| - Eukaryotic = oestrogen responsive transcription
204
Transcriptional switches
| give a tissue specific and a complex regulatory circuit example
- Tissue specific transcription= beta globin
| - Complex regulatory circuit = cell cycle
205
describe the lac operon
- Prokaryotic paradigm for regulation of transcription
206
what is the lac operon needed for
- For ecoli to be able to break down lactose
- 3 adjacent genes are regulated in the same way located next to each other
- Lac operons = expressed when there is lactose around
207
describe how the lac operon is needed
- Bacteria = growing w gucose and lactrose and these genes will be inactive
- This works w the epithelial promoter
- Allowing for the effective digestion of lactose
- It’s the interaction between the repressor and the lactose that turns the switch “on”
- CAP protein catabolite activator protein
- Will stabilize polymerase complec and allows copying of complex Z genes
- Bacteria will express lac repressor protein
- Lac repressor = another DNA binding protein
208
When the bacteria run out of glucose.. what happens to the lac operon
(and there is a low [glucose] CAP (catabolite activating protein) is activated
- 1. Bacteria will bind to lactose inhibitor, causing a conformational change
- 2. Making it no longer able to bind to DNA
(catabolite activating protein) is activated
209
what do regulatory factors do and how do they recognise their target sequence
recognize their targets sequence
- They recognize by interacting w the DNA
- They don’t have to unwind the DNA double helix to see their target – can leave it as a double
- Transcription factors will recognize and bind the regulatory element
- Transcription = regulated by the efficiency of recruitment of RNA polymerase 2
- Regulatory factors bind promoters
- Help recruit the general transcription factors
210
How is the cell cycle regulated
- Theres a mitogenic signal
- G1= when cells start to prolifersate
- S phase = Synthesis phase where the DNA starts to copy itself
- G2 = checking cells have been properly regulated
- Changes @ level of gene expression needed
- Insensitive to external environment
211
describe the involvemnt of cdk in the cell cycle
There are cyclin dependent kinases
- Regulate cell cycle transitions
- Cyclin dependent kinases target transcription factors
- The key event between G1 to the S phase is the transcriptional activation of genes
- Genes that encode proteins that are involved in DNA replication
212
give examples of factors that activate promoters for G1/S transition genes
E2F
- For S phase to occur
- Certain genes have to get expressed
- DHFR
- Ribonucleotide reductase
- Thymidine kinase
- DNA polymerase
- prB = retinoblastoma gene
213
when is E2F expressed
- E2F = repressed in G0 and early G1
214
what is E2F a product of
- E2F = repressed in G0 and early G1
| - By the product of retinoblastoma gene
215
what is Rb a common target of
- E2F = repressed in G0 and early G1
| - By the product of retinoblastoma gene
216
in cervical cancer what kind of mutations are there
- Rb = common cancer target
- Rb mutations
- In cervival cancer
- One of the proteins can compete for binding to R8
- Which is good for viruses if the cells are cancerous
- Bc there are are more cells for it to bind to
217
What is chromatin
- Chromatin is a complex of macromolecules found in cells, consisting of DNA, protein and RNA.
218
- The primary functions of chromatin are
1) to package DNA into a smaller volume to fit in the cell, 2) to reinforce the DNA macromolecule to allow mitosis, 3) to prevent DNA damage, and 4) to control gene expression and DNA replication.
219
What is a histone
- Wraps around DNA chunks
| - Protein responsible for the first level of packaging
220
what is the nucelosome
- DNA + histones = the nucleosome
221
give the 4 levels of packaging involved
- 1st level of packaging - nucleosome
- 2nd level – nucleosome packing in to 30nm packing
- 3rd level – 30nm packaging into 80-100nm fibres
- 4th level – mitotic chromosome
- The 4th level is also 10k fold packaging
222
What do chromosomes constist of, mostly?
- DNA
- Histone proteins
- Non histone proteins
- Non coding RNA
223
how is the DNA packaged in nucelosomes in the interphase chromosome
- In the interphase chromosome, almost all of the DNA is package @ 1st level in nucleosomes
- Much DNA gets packed into higher order structures
- Not as highly packed as int the metaphase chromosome
224
The nucleosome is.....
- Histone + DNA
- First level of chromatin packaging
- 2 turns of DNA wrapped around a core
225
how many histones make up the core
- Core made up of 8 histones
| - Histones assembled to make up octamer core
226
what is the histone core made up of
- 2x h2A, 2 x h2B, 2 x h3, 2 x h4
227
what is outside this octamer histone core
- There is a positively charged N terminal tail that is outside this octamer core
228
how many base pairs around the histone core
- In these 2 turns around the histone core there is 167 base pairs
229
detail chromatin extraction and the physiolgical conditions that are involved
- Chromatin = extracted @ physiological saly concentration so lots of it appears @ 30nm thick fibre
- Fibre made up of nucleosomes
- These nucleosomes are tightly packed together
- 2nd layer of packagain = the 30nm fibre getting more tightly compacted
- And this 30nm fibre can get even more tightly packaged to make 80-100m fibres
230
Ingredients needed for the compaction of nucleosomes
- Linker histones
- H2 binding to core histones + linker DNA
- Linkers help the histones to pack together more tightly
- There is interaction of histone tails
- With adjacent nucleosomes
- There is binding of the packing proteins to histone tails
231
is chromatin structure static
- Chromatin structure = not static
232
What happens to nucleosomes during trancription
- During transcription / DNA replication the nucleosomes have to get removed from the DNA in front of polymerase
- RNA molecules take the nucleosomes out of the way
- Nucleosomes are transciently taken off and then put back on in a different position
- There are twice as many histones than whats needed during DNA replication
233
Histone Remodelling factors
- Replace the nucleosome
- When nucelosomes are in the way of RNA molecule & ar ATP dependent
- ATP dependent – require energy to pick up and drop the molecules
234
Euchromatin (“with activity”) (euros)
- Lightly staining areas of chromatin that are GENE RICH
- Euchromatin = made up of nucelosomes
- But not dense and higher order packaging
- Euchromatin = where the active genes are
235
Heterochromatin
Has few genes
... give some more info
- Darkly staining areas of chromatin
- Heterochromatin has few genes
- With dense higher order packaging of nucleosomes
- Heterochromatin – inactive DNA1
236
Facultative heterochromatin
- Contains all genes not expressed in cell type
- But DNA packed as tightly as heterochromatin
- But it can be packaged as euchromatin in other cell types
- Packed as euchromatin where the gene is acted for expression
- There are factors that can control whether the nucleosomes get packed as euchromatin (active genes)
- Or heterochromatin (inactive gene)
237
What are the key levels of control
- Chemical modification of lyseine residues
- In the histone tails
- Via acetylation and methylation
- There are other levels of structure in chromatin.
- You can treat chromosomes to extract histones + most non histone proteins
- They do not completely fall apart when you pull out the histone core
- They will just appear as long DNA loop
- Which are attached as a scaffold of tightly bound proteins
- The loops of DNA emerge from the scaffolding
- These scaffold form domains and there is insulation
238
Loops and chromatin domains ... describe them
- Each loop has a different degree of chromatin comparison
- The scaffold isolates the chromatin in 1 loop from next loop (the horizonal bit that goes across)
- One loop might open up the chromatin and activate genes
- Neighbouring loop can be tightly packed as heterochromatin
239
where does DNAse digestion occur
- DNAase 1 sensitive sites (HSS)
240
do some DNA sequences have histones
- Can have naked DNA
- Or could be transcription factors that bind these
- Are cut by brief digestion with DNAs1
- Found in promoters and enhancers
241
is there a certain place that enhancers are
- Enhancers can be far away from the promoter
- They can be a long way up or downstream
- Key sites with no histones or nucleosomes
242
ChIP
| Chromatin immunoprecipitation - what is it
breaking of these ChIPs into smaller fragments
- Breaking chromatin into small fragments
- They smash up the cell in some way to get the outside extract of the cell
- An antibody gets added to the target protein
243
- Immunoprecipitation precipitate contains what
- Immunoprecipitation precipitate contains a 1. Target protein 2. Associated protein 3. DNA
ChIP
Chromatin immunoprecipitation
- breaking of these ChIPs into smaller fragments
- smash cell
- outside extract - Breaking chromatin into small fragments
- They smash up the cell in some way to get the outside extract of the cell
- An antibody gets added to the target protein
Antibodies
- GATA 1 (transcription factor)
- Acetylated histones
- Immunoprecipitation precipitate contains a 1. Target protein 2. Associated protein 3. DNA
- Protein that we want is then isolated – antibodies can recognize their antigens
- In precipitiate globin genes are particularly enriched
244
Regulatory transcription factors - what do they do
- Binding occurs just upstream to TATA box (5’ end)
- General transcription factors recruited
- An initiation complex is assembled
- And RNA polymerase gets recruited
245
what is involved in cel differention / expression and senescence
- There are memory control circuits in differentiation and determination
- There are master gene regulations
- And positive feedback
- Such as melanocytes and muscle
246
What is cell senscence
- Biological ageing
- Important in cancer and ageing stem cells
- In somatic cells
247
What are the mechanisms for memory
- Distinguishing factor of cell differentiation = memory
- When cells differentiate it can remember this state
- Can remember without any external signal
This is through chromatin remodeling and positive feedback
- Cells can remember what they are supposed to be
248
what happens in the mechanisms of chromatin remodelling
- States of DNA methylation and histone modification
| - States can be copied to daughter cells
249
what is Positive feedback in relation to cell sensecence = memory
- Feed forward
- The process of keeping something going once it has started already
- Signal causes A to appear
- Signal = something like a hormone or growth factor
- When signal goes away then this remains as a stable thing & the scheme is set up
- B causes A to be made and A produces B
250
melanocytes mechanism of memory involving positive feedback
- MITF = their master gene regulator
| - This is a transcription factor that coordinates many or all of the specialized subtypes
251
skeletal muscle mechanism of memory involving positive feedback
- MYOD1 family
252
What is the master gene regulator
- Transcription factor
- That coordinately regulates many or all of the specialized genes
- That are expressed by a particular cell type
253
Positive feedback in melanocytes to
| Differentiation of the melanocytes. what is the mechanism
- MITF is their master gene regulator
- (Melancocyte I transcription factor) to remember, but this acc stands for the microphalmia associated transcription factor
- Here the signal is MSHMC1R this produces cAMP which produes MITF as B.
- When the MSHMC1R signal is removed the cAMP will continue to produce the MITF.
- The MITF backward reaction produces cAMP.
254
MITF (what is produced by the cAMP when the signal of MSHMC1R is applied to melanocytes in differentiation) --> what happens in them
- Severe mutations in MITF gene
- When its homozygoud
- This causes the loss of all melanocytes in the body
- Eyes can get smaller
- There will be a loss of the pigmented retine
- Microphtalmia = micro opthalmia ]]
- We need MITF for all normal melanocyte development
- + = normal gene
- - = mutant gene
- - / - = both copies are mutant
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What is waardenburg syndrome + what does it result from
- This results from a mutation
- There is a mutation of one copy of the MITF gene in humans
- Can cause deafness and congenital patchy loss of pigment in the skin, can include viruses
- The human eye = w/ no melanin = light blue
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So how goes the MSH-MC1R complex start the production of cAMP, and thus MITF in the first place?
- MSH binds to the MC1R receptor
- Through Gs pathway will activate adenylate cyclase
- This will then stimulate the production of c AMP, which will then produce pKa
- Then this pKa will act on phosphorylated CREB and activates it, causing it to undergo transcription and produce genes including MITF
- MC1R has some basal activity so this process can be stimulated even if there is no MSH present
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Another parallel in the positive feedback of melanocytes differentiation (not the MC1R one)
- SCF can produce KIT
- KIT will then stimulate MAPK, which makes MITF
- MITF makes more KIT
- KIT = receptor w tyrosine kinase activity
- Ligand stem cell gactor that is found in the skin
- This only works when SCF is present
- KIT doesn’t have any basal activity like the MC1R
- Therefore can only activate MAPK and upregulate MITF when SCF is present
- Melanocytes migrate to different parts of the body if there is no SCF present
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Skeletal muscle differentiation feedback- Myogenic factors : what do they do and how do they work
- - MYOD 1
- MYF5
- MYOGENIN
- MRF4
- Bind and activate muscle gene promoters
- They work as dimers with E proteins
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E proteins ? are they
- Are widely expressed transcription factor
| - Myogenic factors normally as dimers with E proteins
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ID1 stands for what and what does it do
- Inhibitor of Differentiation 1
- A proteins in myoblasts
- Can strongly bind E proteins but cannot bind DNA
- (ID strongly binds E proteins)
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what do the myoblasts have At low activities
- At low activities the myoblasts have MYOD factors
- Where they differentiate and have less growth factor
- Leaves destabilization and myogenic factor fere to bind
- All activate the muscle genes
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Cell senescence
| - Major defence against what
cancer
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cell senscence is what
- Cell senscnence = permanent cell growth arreast
- Following extended cell proliferation
- Senescence happens after the cells have divided many times and normal diploid cells will stop dividing
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how many divsions do fibroblasts have before they die
- Fibroblasts for example gets to a total of 50 cell divisions before they die
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- Replicative senescence is what
is the permenant cell growth arrest following extended cell growth proliferation
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what is the Hayflick limit
fact that Cells have a finite lifespan
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Morphological changes - Eg in senescent human fibroblasts : what molecule markers do senscent cells have
- Lysosomal Beta galactosidase
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give an example of a cell cycle inhibitor
- P16
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what are telomere
- Telomeres are 1000s of repeats
- Of a hexamer sequence @ chromosome ends
- The hexamer sequence @ end of the chromosomes is TTAGGG
- The 3’ ends of linear DNA (downstream)
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can telomeres usually be replicated
- Cannot be normally replicated
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what is needed to maintain the length of telomeres
- Telomerase enzyme is needed to maintain the length of telomeres
- Telomeres are able to replicate the telomerase length
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Telomerase
- Protein – RNA complex
| - Telomerase enzyme = able to replicate telomeric DNA
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how does telomerase replicate telomeres DNA
- It does this by reverse transcribing the DNA hexamers (TTAGGG) from its own RNA sequence
- It then joins them to the chromosome end
- Bc its repetitive it is able to add as many as we want, without adversely affecting function
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where is telomerase activity the highest
- Telomerase actually is highest in germ cells that are along normal cells
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what has the longest telomeres
- Germ cells = have longest telomeres
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how long are germ cells in humans
- Germ cells = about 15kb in humans
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what are TERT and TERC
```
TERT = Telomerase reverse trancriptase
TERC = Telomerase RNA Component
```
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how much TERT do most human somatic cells express
- no TERT
| - Therefore there is no telomerase activity
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what happens to telomeres as the cells divide
- Telomeres shorten as the cells divide
- So the last bit of DNA cannot be added on
- Once the telomeres get quite short (almost no telomere left @ all)
- When theres little telomere left
- Cell senescence = stimulated
- Shortness of it will get replicated
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What is the link between telomeres, cell senescence and cancer
- Cell cancer lives in culture
- Nearly all of the cells (90% ish) will express telomerase reverse transcriptase
- By expressing TERT they are immortal
- Cell senescence is an important barrier for cancer
- Cancer cells have found a way to turn TERT back on
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explain telomeres and senscene principles
- When a few telomeres get to a short length then the telomeres will stop dividing
- The illustrates the senescent culture
- GGG + SPERM ZGOTE (goes to somatic cells)
- Somatic cells are not generally immortal
- There is no telomerase
- Normal germline cells (oocytes/sperm and their diploid progenitore), they DO express TERT
- Expression of TERT means that full length telomeres
- The germline is immortal and can divide forever
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is loss of TERT good?
- Loss of TERT is good ; in a way & can stop development of cancer
- If cells stop growing you get a tiny lesion that is not cancerous
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P53 tumour suppressor .. describe it
- Tumour suppression
- Cell thinks that there is a piece of broken or damaged DNA
- P35 turns on
- P53 will stop further cell division
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P16P16
- Inhibits CDK4 and CDK6
- Some cells switch on these pathways
- And some cells turn on other pathways
- Advanced cancer cells have usually bypassed cell senescence.
- Some of the most common abnormalities that are found in cancer cells
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problems in the cancer cells
- 1. Defective senescence
- 2. Immortality
- Includes the expression of TERT
- P53 defects and p16 defects
- Cancer cells can get around this
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Telomere length with age
- Gets shorter w age
- But its variable within people
- Can be measured in blood cells
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P16 tumour suppressor
- Expressed more with increasingly ageing tissues
- Other senescence associated proteins
- CDKN2A locus (P16) is genetically associated with human senile defects
- Such as CV disease / frailty/ DM2/ Cancer/ neurodegeneration
- Telomere length @ birth is genetically linked to age and death
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- A stem cell is one that is capable of
- 1. Self replication dividing into more cells like itself
- 2. Differentiation into one + kinds of specialized and functional cells
- Can be unipotent and only form one functional cell type
- Pluripotent and makes several functional cell types
- Totipotent can form all functional cell types
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zgotes characteristics
- Are totipotent
- Not usually considered a stem cell
- It doesn’t divide to make more zygotes
- Cells of inner cell mass of an early mammalian embryo can act as totipotent stem cells
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give eg of zygotes
- Embryonic stem cells
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Embryonic stem cells.... what their crack
- Express TERT (telomere reverse trancriptase )
- Therefore are natural immortal because their telomeres cannot get shorter and trigger tumour suppression / growth arrest
- ES cells = considered germ line cells
- Because they can form all cell types, including gametes
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Somatic stem cells exmaple
- Somatic cells
| - Eg postnatal stem cells
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Somatic stem cells telomerase activity
- Remain as proliferative reservoir after birth, in the gut / skin / bone marrow
- Som adult somatic stem cells have some telomerase activity
- But don’t have enough telomerase activity to make a normal adult stem cell immortal
- So telomeres shorten LESS per division in a somatic stem cell than other somatic cells
- But they still do shorten
- Somatic stem cells do gradually senesce
- Children can also have somatic stem cells
- There are connections between stem cell ageing and our ageing.
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Bone marrow as we age.. what happens
- Fall in mortality of elderly
- Increase in bone marrow failure
- Fall in success rate as bone marrow donors
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Hair greying.. why does this happen
- Due to decrease melanocyte stem cell maintenance in hair follicles
- Of the skin
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Skin as we age....
- Fall in healing ability
| - Increased risk of skin illness
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Dermal fibroblasts.. do they have sensence
- Have senescence
- Epidermal stem cells have very little telomere shortening
- And they remain able to divde
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Epidermal cells
- Epidermal cells are some of the immortal cells that we have in the body
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cancer cell differentiation
- Cancer cells are usually poorly differentiated
- Neuroblastoma, retinoblastoma
- Cancer cells = also tend to have telomerase (like stem cells)
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Prostatic cancer
- Cancer stem cells in prostate
- Proposed to be cells with CDI33 and other markers
- A very small and slow growing population (subpopulation)
- But these markers correlate with the ability to form large cell colonies
- Can also form tumours if grafted onto animals
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Cancer stem cells
- This is hypothetical for many cnacer types
- These are a small subpopulation in cancer that rarely divide
- Only cells in the tumour that are immortal
- Only cells that cacn form large clones
- And can regenerate a tumour after depletion
- If there is a rare division; such cells can evade standard therapies
- Because therapies target proliferating cells
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Melanomas
- Alteast 25% of single human melanoma cells
- Can form tumours on grafting to immunodeficient make
- Hence are stem cell like
- No special small stem cell population here
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?is Genetics
- Inheritance of genes
- That are activated by mutation
- Change in DNA sequence
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?is Epigenetics
- Inheritance of a state of gene activation
- Inactivation without any change in DNA sequence
- Active/ inactive chromatin structure is “remembered”
- Its not lost during DNA replication and mitosis
- -> conserved
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Mechanisms of epigenetis and gene regulation
- The nucelosomes get removed in FRONT of the replication fork
- Nucleosome gets replaced behind the replication fork
- There is removal from in front of the replication fork and then its put behind the fork
- New nucleosomes get added
- Because there is twice as much DNA
- There is addition of new nucleosomes because there is twice as much DNA
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what do histones carry
- Histones carry histone markers
| - Such as methylation and acetylation
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what do histones recruit
- HATs + HDACs etc are recruited and add similar marks
| - To new histones
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Mechanism 2: DNA methylation
- Maintenance methylase
- Binds to Hemi methylated DNA
- And then it methylated the unmethylated CG
- The histone modifications and DNA methylation can interact
- This is all a form of positive feedback
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Carcinogens need what
- Need mutations
- In 1. Dominant oncogenes
- 2. Tumour suppressor genes
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Tumour suppressor genes need what
- These need both copied of the Gene in a diploid cell
- To get inactivated
- To allow the cancer to develop
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Tumour suppressor genes
- Cell cycle checkpoint genes eg RB/ p53
- DNA repair genes
- Genes that regulate cell death
- P21 WAF- important cell cycle regulator and inhibits cell cycle progression
- P21WAF acts as TSG
- P21 is inactivated in a lot of cancers
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G2 checkpoint
- P53phosphoryated p53p21cell cycle arrest
- Usually p53 is quickly degraded and unstable
- And is maintained at very low levels
- Phosphorylated, activated p53 is not degraded - The active p53 promotes transcription of genes
- That induce cell cycle arrest (because it’s a tumour suppressant gene)
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P53 tumour suppressor does what
- The active p53 promotes transcription of genes
- That induce cell cycle arrest (because it’s a tumour suppressant gene)
- Active p53 can bind to promoter region of the p21
- And stimulate p21 expression
- P21 will bind and inactivate g1/s-cdk and s-cdk complexes
- Cell arrest in G1
- This allows time to repair damaged DNA
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P21 tumour suppressor does what
- In some malignancies
- P21 is not mutated, but is still inactivated
- P21 gene is packaged as condensed heterochromatin
- This is called epigenetic inactivation
- The condensation is maintained via repeated cell divisions keep dividing the condensed ones so that it remainds condensed.
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HDAC inhibitors --> give examples
- Volinstat
- Romidepsin
- Panobinostat
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- Treatment of tumour cells shows what
1. Reactivation of p21
2. Apoptosis
3. Cell cycle arrest
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Sickle cell disease symptoms
- Presentation of pain crises, infection, anaemia
- Organ chrises (lung/spleen), chronic organ failure (kidney)
- This happens due to a point mutation in the beta globin gene
318
The point mutation that occurs in sickle cell disease is what
GLU --> VAL
| GAG to GTG
319
VAL is what
- Neutral hydrophobic amino acid residue
320
HDAC inhibitors examples
butyrases
321
DMT inhibitors examples
decitabine
322
Hydroxycarbamide and how it works
used clinically
- Hydroxycarbamine induces HbF
- This induction of HbF gives som protection from the symptoms of sickle cell disease
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DNA microarray method
DNA microarray method
324
Next gen DNA sequencing
- Massively parallel DNA sequencing
- 50k + DNA molecules are sequenced @ same time
- Several different technologies are used
- DNA molecules can be trapped on a chip
- A sequencing reaction is performed w fluorescent nucleotides
- The chip can be scanned with a microarray scanner
- The sequence for each DNA molecule is read.
This is the DNA molecules that are trapped on a chip
- Chip after having been scanned with a microarray scanner
- The sequence for each DNA sequence is read
325
Desmosomes are.....
molecular complexes of cell adhesion proteins
326
What do desmosomes do
- link proteins that attach the cell surface adhesion proteins
- to intracellular keratin cytoskeletal filaments.
327
Desmosomes are.....
molecular complexes of cell adhesion proteins
328
What do desmosomes do
- link proteins that attach the cell surface adhesion proteins
- to intracellular keratin cytoskeletal filaments.
