BMS238 Cell and Molecular Biology Flashcards
Why is protein folding required?
For acquisition of the correct protein structure to perform designated function
What can protein misfolding lead to?
the failure to acquire the correct structure hence the failure of function
Give some examples of diseases that are caused by the protein misfoldings?
Huntingtons, Cystic fibroisis, Parkisons, Alzheimers
Why can researching protein structure be difficult?
Because proteins are made up of 20 different amino acids and the average protein has 500
- Each amino acid has its own complex structure e.g. alanine has 12 atoms
- So simplifying structural features allows easier understanding
What are the two main secondary structures of protein?
Alpha helix
Beta sheet
What are the different ways in which you can represent secondary protein structure?
- Backbone diagram
- Sticks (shows side chains)
- Space filling (shows all the atoms)
- Ribbon (follows the path of the backbone) - most useful
What is the structure of a beta sheet?
- Beta sheets consist of beta strands connected laterally on adjacent regions of the peptide backbone by hydrogen bonds. These interactions do not involve side chains
- The side chains extend above and below the beta strands allowing one side of the sheet to have different properties to the other
How are beta sheets usually represented?
As flat arrows pointing in the direction of the c terminus
- Central strands run parallel
- Peripheral strands run antiparallel
What is the structure of an alpha helix?
A spiral confirmation a N-H group from the backbone of an amino acid donates a hydrogen bond to the backbone C=O group of another amino acid located four residues further along the protein sequence. These interactions do not involve side chains
What amino acids do not favour alpha helix formation?
Proline and glycine
How has evolution fine tuned protein function?
By adding functional domains
What ‘interactions’ hold proteins together?
Hydrogen bonds
Electrostatic
Van der valls
Disulphide bonds
What are hydrogen bonds?
Involve a H shared between O and N atoms
What are electrostatic interactions?
Attraction between +ve and -ve charged ions
What are van der walls?
short-range hydrophobic interactions
What are disulphide bonds?
Involve a chemical link between two adjacent cysteines
What decides how a protein folds?
Hydrophobic and polar side chains
- Polar side chains form hydrogen bonds with water meaning they are on the outside of the protein to allow for reactions to occur
- Hydrophobic side chains are in the middle - repelling water forcing the protein to hold its shape
What are the levels of protein structure?
Primary - polypeptide chain
Secondary - alpha helix and beta sheets
Tertiary - whole protein
Quaternary - Several proteins
What is tertiary structure of a protein?
The way in which individual secondary structural elements; alpha-helices, beta-sheets and random coil, pack together within one protein
What is the quaternary structure of a protein?
Quaternary structure is the relationship between individual proteins in a multimeric complex (often duplication and multimersiation of subunits occurs).
Protein is said to contain subunits.
How do we find the primary structure of a protein?
Most primary structure is inferred from DNA sequence
But it can be determined directly by amino acid sequencing using Edman degradation or using mass spectrometry
What is Edman degradation?
- Phenyl isothiocyanate (PCT) is used to break away the terminal amino acid
- You can then determine the unique sequence by high performance liquid chromatography (HPLC). This is done based on the molecular weight of each residue
- Each amino acid has its own molecular weight, can therefore work out which amino acid is being removed each time and subsequently the primary structure
How can the primary structure of a protein be used to predict the secondary structure?
- By aligning it with other similar proteins (similar sequences of amino acids) with known structures
- Also some amino acids need to be in certain positions for structure to develop
What does amphipathic mean?
A polypeptide chain with both hydrophobic and polar side chains
How can alpha helices interact with each other?
Double or triple coiled coil
- Coil together alpha helices with their hydrophobic side chains facing inwards creating a hydrophobic core
Where are triple coiled coils found?
In elongated fibrous proteins that need the ability to form cloths
What techniques can be used to find real protein structure?
Circular Dichroism (CD)
X-ray Crystallography
Nuclear Magnetic Resonance (NMR)
Electron Microscopy
What is required for the techniques that are used to find real protein structure?
You need to purify the protein of interest
into its purest form.
What is Circular Dichroism used for?
To estimate the secondary structure of a protein?
How does circular dichroism (CD) work?
- CD spectroscopy uses far-UV radiation (190-250nm)
- Each secondary structure (alpha helix, beta sheet, random coil) give a characteristic shape on the CD spectrum. The fraction of the protein in each secondary structure can therefore be calculated from the CD spectrum, producing a percentage of each secondary structure present in the protein
- Α helices have two-peak spectrums at 208 and 225nm whereas, β sheets give a single peak between 216-218nm. Random coils also have a unique CD profile
How can circular dichroism be used to show protein stability?
Can be used at different temperatures providing us with information regarding the proteins stability – the less stable, the quicker the structure will be lost
What is calmodulin?
Calmodulin is a small calcium-binding protein which activates protein kinases and other proteins
Where are the binding sites on calmodulin?
There are two globular domains each with two calcium binding sites - total of four
Sites occur in the loop region connecting two alpha helices
What happens when 4 Ca2+ ions bind to calmodulin?
Allosteric changes
- The two globular domains would rotate allowing calmodulin to bind to target proteins and regulate their activity
- The central helix of calmodulin breaks into two allowing calmodulin to wrap around helical peptides on target proteins
What is NMR used for?
NMR spectroscopy is useful to understand dynamics of protein structures
What is requires of the protein of interest in NMR spectroscopy?
The protein of interest needs to be highly pure and labelled with isotopes such as 13C or 15N
How does NMR spectroscopy work?
- The subatomic particles of the isotopes possess a quantum-mechanical spin. These spin vectors are aligned with a large magnetic field in several configurations determined by energy state
- Radiowaves are then used to resonate with the natural frequency of these particles spins and cause a transition in spin vector orientation to a high-energy conformation.
- The NMR machine then records the different frequencies required for resonance to occur. This attribute, known as chemical shift, is dependent on the local environment and can be used to determine protein structure
How can you label a protein with an isotope (as required in NMR)?
The protein must be highly pure so is produced recombinantly in bacteria that have been grown on a media where the sole nutrient source is 13C or 15N. This will produce the protein labelled with these isotopes
What is x ray crystallography?
A high energy and focussed beam of X-rays is fired through a protein crystal. Most of the X-rays go straight through, but a few are deflected giving rise to a diffraction pattern. The structure of the protein is effectively traced back from the diffraction pattern using modern computational techniques
What are the two type of electron microscopy used to study protein structure?
Transmission electron microscopy
Cryo-Electron microscopy
What is transmission electron microscopy used for?
To study large protein structures
How is transmission electron microscopy used to study protein structure?
The protein of interest is placed on an EM grid and spiked with a solution containing a heavy metal shading that is impermeable to electrons
Electrons are then fired at the protein and a negative stain is produced
Image analysis is then employed to build-up an average structure
How is Cryo-Electron microscopy used to study protein structure?
- Cryo-EM consists of using liquid nitrogen to preserve/freeze the protein specimen
- Electrons are then shone onto the frozen specimen and the average shape of the protein is determined
- The more ordered and symmetrical the protein specimen is that is used in cryo-EM, the easier the averaging process
What are the pros and cons for using circular dichroism to study protein structure?
- Cheap, Quick, no size limit
- Limited dynamics, low resolution, secondary structure only
What are the pros and cons for using X ray crystallography to study protein structure?
- High resolution, no size limit
- Expensive, slows down due to crystal formation
What are the pros and cons for using NMR to study protein structure?
- Reasonable resolution, good dynamics
- Size limit of 50kDa
What are the pros and cons for using electron microscopy to study protein structure?
- No size limit - large structures
- Quite expensive, medium speed
- No dynamics
Why do we need to purify proteins?
Because cells contain 20000-30000 proteins
We therefore need to know the structure and function for each individual protein
Even secretary fluids have a large mix of proteins
Give the steps in protein purification
- Tissue homogenisation by sonication, blending, pestle and mortar
- Separation of the released material from unbroken material by centrifugation
- Several chromatography steps
- Confirmation of protein purity by electrophoresis
- Confirmation of protein identity by Western immunoblotting
- Confirmation of protein identity by mass-spectrometry
What are the steps in differential centrifugation?
Spin the cell homogenate at a low speed
- pellet containing whole cells, nuclei, cytoskeletons
Spin the supernatant at a medium stage
- Pellet contains mitochondria, lysosomes and peroxisomes
Spin at a high speed
- Pellet contains microsomes and small vesicles
Spin at a very high speed
- Contains ribosomes, viruses, large macromolecules
- Pure cytosol
What are the two types of density based ultracentrifugation?
Velocity sedimentation
Equilibrium sedimentation
What is velocity sedimentation ultracentrifugation?
- A test tube containing a stabilising gradual sucrose gradient is established (highest concentration at the bottom)
- Addition and centrifugation of the cytosol separates components based on density
- A hole is punctured in the bottom of the test tube and organelles can be collected from the bottom in various fractions depending on their density
- Heavier organelles will sediment quicker and lighter ones will take longer
What is equilibrium sedimentation ultracentrifugation?
- A steep sucrose gradient is established in a test tube with highest concentrations at the bottom
- The cell contents are then added and centrifuged for a long time at a very high speed
- The organelles deposit in the sucrose depending on their density with heavier, denser organelles depositing at the bottom of the tube and so on
What is gel filtration chromatography?
- Cytosol that has previously been centrifuged is loaded into a column containing a solid matrix of beads of a certain size
- Neutral buffer and solvent added to push cytosol down column
- Larger proteins have a smaller volume to move through, due to their size. Larger proteins leave the column first
- The different fractions collected drop-by-drop over time
How is the working range of the resin used in gel filtration chromatography defined?
The resin used has a working range which is defined by the pore size of the beads. If proteins are too large to fit through the pores, they are excluded
What is affinity chromatography?
Relies on the tight interactions of enzyme-substrate binding and allows separation of specific substrate binding proteins from the cytosol. Bound protein is eluted using a competing ligand or high salt concentrations
How do you purify specific DNA binding protein by affinity chromatography?
- Cytosol added to column containing beads with a covalently attached substrate
- Enzymes for the substrate bound to the bead bind irreversibly if the substrate is non-hydrolysable
- Other proteins in the cytosol will pass straight through the column and only the enzyme will be retained
- Elute the protein of interest using salt
What is ion exchange chromatography?
- Diethylaminoethyl (DEAE) beads added to a column in addition to negatively charged proteins
- The proteins will bind to the positively charged DEAE beads
- An increasing concentration of salt is then added to the column to displace the proteins form the beads
What solvents do you use in ion exchange chromatography?
Cation exchange
- CM carboyx-methyl
Anion exchange
- DEAE diethylaminoethyl
How do you remove the proteins from ion exchange chromatography column?
- Less negatively charged proteins are displaced and release from the column first, at lower salt concentrations
- More negatively charged proteins will be displaced later at higher salt concentrations. This allows you to separate fractions based on charge
What is meant by the three stem protein purification?
Using a mixture of ion exchange, gel filtration and affinity chromatography
What is SDS-PAGE?
Sodium dodecyl sulphate polyacrulamide gel electrophersis
Outline SDS-PAGE
- Proteins are boiled and SDS added. SDS is negatively charged and hydrophobic. Proteins fold hiding the hydrophobic centre. Proteins can then unfold after the addition of the hydrophobic SDS forming a stick allowing them to move easily through the gel. SDS makes the proteins have an equal charge to mass ratio
- Proteins are placed in a well behind the gel and a current is applied
- The negatively charged proteins move towards positive anode
- Smaller proteins will move faster and hence further through gel whereas larger proteins will move much slower
Why are proteins essentially sorted by molecular weight not charge in SDS-PAGE?
- SDS coats the proteins with a uniform negative charge, masking their charges
- SDS binds fairly uniformly to the linear proteins meaning that the charge of the protein is now approximately proportional to its molecular weight
- This means that there will be now differential migration based on charge so larger proteins will have the same attraction to the anode as smaller proteins but larger will travel slower as they can’t pass through the gel is easily
What is the role of mercaptoethanol?
Denatures the protein structure due to its CH group
Breaks down polypeptide chains into smaller fractions which can then be investigated separately
What is 2 dimensional gel electrophoresis?
Used for complex samples
1st dimension
- A stable pH gradient is created in a commercially available gel. Proteins are introduced to the gel and run along it until they reach the pH that corresponds to their isoelectric point
- At this pH, the proteins become uncharged and no longer run along the gel causing the proteins to separate by their native charge
- Commonly used in proteomics and in complex samples
What is western blotting?
- Separated proteins are transferred to a membrane using electrical current
- Excess of primary antibody is added to the membrane which binds to target protein
- After the unbound antibody is washed off, an excess of a secondary antibody is then applied to the membrane
- Secondary antibody binds selectively to the Fc region of the primary antibody
- It is tagged with an enzyme which when activated leads to the production of a fluorescent or coloured product
Why must we use two antibodies in western blotting?
It would be too expensive to label each primary antibody with an enzyme as there is too many
Instead use secondary antibody that can detect the primary ones
Secondary are only used to determine location of unlabelled primary antibody
How can you detect the antibody binding in western blotting?
H202 addition produces lots of light can look via X Ray
What is mass spectrometry?
Allows us to identify the labelled peptide
- Isolated protein incubated with protease (trypsin or peptitrypsin) to digest it into peptides
- The small peptides are then run in mass spectrometer which ionises the peptides - You can then identify specific peptides using its database of known peptides molecular weights to compare with
Why is trypsin used in mass spectrometry?
Because only small polypeptide chains can be put into the mass spectrometer
- Trypsin cleaves the polypeptide chain after lysine
- Lysine has an abidance of 6% in humans so is likely to result in a small chain
Trypsin is also cheap
Why is mass spectrometry useful?
Because it allows identification and sequencing of tiny changes in proteins
How does a molecule being labelled by a phosphate affect its mass spect result?
The presence of a P04 group increases the mass of serine by 95 daltons so a fraction will change if labelled with a phosphatase
Which amino acids can be phosphorylated?
Serine
Threonine
Trysoine
Because they have hydroxyl groups
Define proteomics
The term Proteomics refers to the analysis of complete protein content in a living system, including post-translationally modified proteins and alternatively spliced variants
Why is mass spectrometry crucial for proteomic studies?
Mass Spectrometry has now become a crucial technique for almost all proteomics experiments. It allows precise determination of the molecular mass of peptides as well as their sequences. This information can very well be used for protein identification, de novo sequencing, and identification of post-translational modifications.
Name some post translational modifications of amino acids
Hydroxylation
Methylation
Acetylation
Lipids
How does hydroxylation change an amino acid?
Adds an OH group
+17 Daltons
How does methylation change an amino acid?
Addition of CH3 group
+15 Daltons
Has an important role in epigenetics
How does acetylation change an amino acid?
Addition of CH2CH3 group
+27 Daltons
Role in epigenetics
How do lipids change an amino acid?
Varies depending on the modification
- Usually +2000 Daltons and higher
When were chromosomes discovered?
1902 by Boveri and Sutton
Morgan discovered that chromosomes are the locations of genes in 1915
What is contained in chromosomes?
DNA and proteins
What are the functions of the proteins in chromosomes?
- Packaging and unfolding of DNA within the nucleus
- Controlling DNA replication, DNA repair, genetic recombination
- Maintaining chromosome integrity by preventing loss of end sequences
- Governing proper chromosome segregation during cell division
- Regulating gene expression
Are chromosomes only found in the nucleus?
No small circular chromosomes are found in mitochondria and chloroplasts
At what stage in the cell cycle are chromosomes easily distinguished?
Metaphase
Define karyotype
The organised representation of all chromosomes in a eukaryotic cell and metaphase
How can different chromosomes be distinguished from each other?
Chromosome painting
- Allows chromosomes to be distinguished based on its DNA sequence content
- This technique is often used in place of measuring chromosomes
Measuring chromosomes
- Each pair of chromosomes are different sizes
How are chromosomes arranged in a nucleus undergoing interphase?
Normal running of cell - housekeeping functions
Can see that each chromosome occupies a distinct subnuclear territory in the interphase nucleus
How does the location of transcriptionally active and inert genes differ in a nucleus?
Active genes are in the centre and inactive are in the peripheral
There are some exceptions but this is generally the pattern
How was the location of active and inert genes investigated?
Gene specific paint was used to investigate the position of a signal responsive gene
- When active the genes (one on each chromosome) can be seen in the centre of the nucleus
- When inactive the genes were pushed towards the periphery
What is a chromosome?
A highly coiled fibre of chromatin
What is seen if you look at an interphase chromatin under an electron microscope?
Resembles beads on a string where the beads are nucleosomes
What is the structure of a chromatin fibre?
It is a supercoiled array of nucleosomes
What is a nucleosome?
DNA wrapped around a protein core
Describe the protein core of a nucleosome
Consists of 8 subunits called histones
- There are 4 types of histones, each with two copes
- The N terminal tails of the histones project out of the nucleosome and are free to interact with other proteins facilitating regulation of chromatin structure and function
What is the role of histone H1?
It is a linker histone that straps DNA onto histone octamers and its the movement if DNA relative to the histone octamer
- Important in chromatin condensation process which shrinks interphase chromosomes to metaphase size in cell division
What is the structure of the N terminal tail of histone molecules?
Rich in basic amino acids such as lysine
How can remodelling of chromosome structure occur?
Axillary proteins strip off DNA by removing histone H1
This allows the proteins to displace the histone core away from the DNA that is it is usually bound to
Produces a stretch of nucleosome free DNA which can be readily transcribed - accessible by transcription factors
Why do chromosomes need to contain specialised DNA sequences?
Because the sequences facilitate reliable and complete DNA replication, segregation of duplicated chromosomes during cell division
What is a telomere?
DNA sequences at ends of linear chromosomes: maintain chromosomal integrity
What is a replication origin?
DNA sequence where DNA replication is initiated
What is a centromere?
DNA sequences on which kinetochore assembles and mediates chromosome segregation at mitosis and meiosis
What is a kinetochore?
Protein complex that binds microtubules in the mitotic spindle
What is the role of a telomere?
A specialised DNA polymerase called telomerase produces a single stranded 3” overhang repeat called a telomere
- Repeat is TTAGGG
- Added after replication and stops the removal of DNA nucleotides at the end of the DNA strand
Where are centromere seen?
In the centre of the nucleus at the end of microtubule attachments
What do centromeres consist of?
Alpha satellite DNA repeats which form condensed heterochromatin with histone octamers containing unusual subunits
How does the kinetochore have a role in cell division?
Kinetochore inner plate proteins bind to alpha satellite DNA
Kinetochore outer plate proteins bind with microtubules (component of the mitotic spindle)
This arrangement occurs on both sister chromatids so that when division occurs the they are pulled in opposite directions
What is different about the structure of the nucleosome in centromere chromatins to normal DNA chromatins?
They have a centromere specific histone 3 variation called CENP-A that is only found in the centromere chromatin and is responsible for the physical interactions with the inner plate proteins in the kinetochore
What is the role of chromatin containing normal histone H3 in the centromere?
Ensure the right pairings of sister chromatins - allow them to be next to each other
How do kinetochore interactions differ in yeast to animals?
In yeast the kinetochore is a basket that links a single nucleosome of centromeric chromatin to a single microtubule
What is the eukaryotic genome made up of?
1.5% protein encoding
50% repeated DNA sequences
20% introns
30% are non of these but are still unique sequences
- Thought to be regulatory and control the activation of the protein encoding genes
What are the repeated DNA sequences of the eukaryotic genome made up of?
A few % DNA transposon
10% retroviral like elements
30% non retroviral polyA rectotransposons
All are genetically mobile and can relocate on the genome
What are DNA transposons?
occupy a few% of the eukaryotic genome
- DNA sequences that are bound by short repeated sequences
How are DNA transposons genetically mobile?
They move by relocation
- Uses transposases in a cut and paste mechanism
- Transposases bundle the transposon up and move it to another location
- The original location is then transposon free and the DNA strand is fixed
How discovered DNA transposons?
Babara Mcclintock - 1952
What are retroviral like elements?
Occupy 10% of eukaryotic genome
- Integrated DNA sequences that transcribe and produce proteins
- These proteins convert RNA copies of the retroviral genome back to double stranded DNA using reverse transcriptase
- This is then inserted back into the genome in a different location
What are non retroviral polyA retrotransponsons?
30% of eukaryotic genome
- Most abdudent type of transposons in the vertebrate genome
- Replicate via RNA intermediate
How do non retroviral polyA retrotransponsons genetically relocate?
Double stranded DNA sequence is transcribed and encoded proteins that have both a endonuclease function and reverse transcriptase function
This leads to the insertion of the newly reverse transcribed DNA into the genome at a different location as the DNA strands have been cleaved by the endonuclease function
What is the Alu gene?
Found in humans and evolved from a dingle gene called 7SL RNA but now has 1000000 copied of this gene
Through the process of non retroviral retrotransposition
What is the mouse homologue of Alu?
B1
How is DNA replicated?
Semi conservative replication
- Each strand is used to make a daughter strand
Which direction can phosphodiesterase binds form?
5’ to 3’
Define 5’ and 3’ end
5’ - Phosphate present on 5’ carbon of the deoxyribose sugar
3’ - OH present on the 3rd carbon
How is a RNA primer extended in DNA replication?
The OH group on the 3’ end carries out a nucleophilic attack on the phosphodiester bond on the incoming deoxyribonucleoside triphosphatase (e.g.cysteine)
- Also produces pyrophosphate
- This stabilises the reaction and makes it effectively irreversible as the breakdown of pyrophosphate by pyrophosphotase (breaks two high energy phosphate bonds) produces lots of energy that is used to form the new phosphodiester bonds
Why is DNA synthesis effectively irreversible?
Because the breakdown of pyrophosphate is extremely exothermic providing the energy needed for DNA synthesis - a coupled reaction
It is therefore effectively irreversible as it would take a lot of energy to be created to match the energy produced by this reaction
What is a replication fork?
Where the DNA strands are separated by DNA Helicase so that DNA synthesis can be initiated
What is the problems with DNA synthesis from the lagging strand?
Replication can only occur in the 5’ to the 3’ direction meaning that the lagging strand cannot continuously be synthesised as its in a 3’ to 5’ direction
How does synthesis of the lagging strand occur in DNA replication?
By synthesising discontinuous fractions called okazaki fragments
Requires contoinous RNA primers to keep synthesising small fragments
- There are short gaps between the Okazaki fragments
- Rionuclease H removes RNA primer creating the gap
- DNA polymerase extends across the gap and DNA ligase covalently links the fragments to make the stand continuous
What is the role of the RNA primer in DNA replication?
DNA primase synthesises a RNA primer from which DNA polymerase can extend from
What is the role of DNA ligase in DNA replication?
DNA Ligase uses the energy of ATP hydrolysis to ligate newly
synthesised, adjacent DNA fragments in a two-step catalytic reaction
- It uses ATP to create a structure where adenosine diphosphate is added to 5’ phosphate facilitating the nucleophilic attack of the 3’ OH of the Okazaki fragments
What is the role of DNA helicase in DNA replication?
DNA Helicase uses ATP to separate parental DNA strands at the
Replication Fork and move the Replication Fork forward
It wraps around and rotates the strand, disrupting the DNA
What can mutations in DNA helicases lead to?
Werner syndrome - Autosomal recessive - Mutation in RECQ helicase gene WRN - Progeria (premature ageing) Bloom syndrome - Loss of function in RECQ family of DNA helices which maintains genome integrity - Rare cancer syndrome
What is processivity in regards to DNA polymerase?
That DNA polymerase is more likely to extend DNA then it is to fall off
-This is greatly enhanced by its association with a sliding clamp which is positioned close to the primer:template junction
What is the sliding clamp?
The sliding clamp is ATP dependant
- The clamp loader and the sliding clamp create a structure that attracts and stabilises DNA polymerase
- It sits on the primer junction
- It looks like DNA helicase and it sits around DNA and spins to help DNA polymerase move forward
- The sliding clamp sits behind and the DNA helicase in front and DNA polymerase in the middle
What is the problem encountered when DNA helicase has separated DNA strands and then moves on?
The single stranded DNA may start to fold back on themselves causing them to become double stranded
How is the re joining of single stranded DNA strands in DNA replication overcome?
Using single stranded binding proteins
- They keep the DNA strands straight to stop them from folding back on its self and becoming doubled stranded
What is the role of DNA Topoisomerases in DNA replication?
DNA topoisomerases prevent DNA from becoming tangled during DNA replication - Unwinding of parental DNA strands at the Replication Fork introduces superhelical tension into the DNA Helix. - Tension is relaxed by DNA Topisomerases, which nick and reseal the backbone of the parental helix
WHat is the difference between type I and type II Topoisomerases?
- Type I Topoisomerases nick and reseal one of the 2 DNA strands, no ATP required - Type II Topoisomerases nick and reseal both DNA strands, ATP required
What is the role of replicators/orgins in DNA replication?
They are specific DNA sequences that direct the initiation of DNA replication by recruiting Replication Initiator proteins
Give an example of a replicator/origin for DNA replication in yeast
Autonomously Replicating Sequence (ARS) Elements
Give an example of a replicator/origin for DNA replication in humans
DNA sequences near to LMNB2, MYC, HBB.
But also defined by chromatin structure (e.g.
nucleosome free region), rather than a specific DNA sequence.
What is required for initiation of DNA replication in eukaryotes?
- Replicator Selection - formation of a pre-Replicative Complex (pre- RC) - occurs in G1 phase
- Origin Activation - unwinding of DNA and recruitment of DNA Polymerase - occurs in S phase
How is it ensured that each chromosomes is only replicated once per cell cycle?
Temporal separation of replicator selection and origin activation events ensures that each origin is used and each chromosome is only replicated exactly once per cell cycle
How is the pre replicative complex (pre-RC) formed?
Occurs in G1
- Origin recognition complex (ORC) binds to replicator sequence
- Activates helicase loading proteins Cdc6 and Cdt1 waging bind to the ORC
- The helicase Mcm2-7 binds as well completing the formation of the pre-RC
How is the activity of pre-RC regulated in DNA replication?
High levels of Cyclin-dependent kinase (Cdk) activity in s phase activates existing pre-RC complexes
Low levels of Cdk in G1 allows the formation of pre-RC formation
What would happen if there wasn’t any telomeres?
Removing RNA primer at the end of the lagging strand by ribonuclease H leaves a gap at the end that cannot be closed by DNA polymerase and ligase leasing to incompletely replicated DNA
If this wasn’t solved then it would lead to progressive shrinkage of chromosomes as ribonuclease H will keep removing RNA primer at 5’ end of lagging strand
How is the progressive shrinkage of DNA strands overcome in DNA replication?
Addition of TTAGGG repeats by telomerase compensates as the loss of telomere sequences caused by RNA primer removal end prevents chromosome shortening
The extended 3’ end DNA is long enough to enable DNA Primase to bind and initiate new RNA primer synthesis which can then be extended as an extra Okazaki fragment
How does telomerase produce a telomere sequence?
Telomerase is a ribonucleoprotein with an intrinsic RNA component that acts as a template on which telomere repeat sequences are synthesised in a step-wise process
– the Telomerase Shuffle
Outline the telomere shuffle
Telomerase extends the 3’ end of the DNA strand by 3 nucleotides, shuffles along and then disengages its RNA
The telomerase then moves along 6 nucleotides and drops down again (base paired with the newly synthesised bases). Then synthesises 6 new bases and shuffles along
This repeats
How do proteins interact with each other?
No chemical bonds are formed, just multiple weak interactions
What is the charge (usually) of a protein DNA binding domain?
They have an overall basic charge (positive) to mediate the interaction with the acidic (negatively charged) DNA strand mostly through interactions with the major groove
What are the classes of DNA binding domains in proteins?
- Leucine zipper motifs
- Zinc fingers
- Basic helix-loop-helix (bHLH),
- β-sheet
How do proteins increase affinity for DNA?
Increase the number of DNA binding motifs they have
What is a leucine zipper domain and how does it bind to DNA?
- Dimers of short coiled-coil sequence and a specific DNA recognition helix
- Two long alpha helices with hydrophobic side chains extending out into space between them (often leucine). These side chains are tightly packed adding stability to the domain
- The side chains extend into the DNA groove to contact bases forming Hydrogen bonds
Give examples of how metal is bound by proteins
Structurally - Zn2+ - Zinc finger
Regulatory - Ca2+ - Calmodulin
Catalytic e.g.. Zinc, iron and copper
What is Zinc finger DNA binding?
- Zinc finger proteins recognise specific DNA sequences
- Tetrahedral shaped by two cysteine residues from the alpha helix and two histidine from the beta sheet
- The helical region of each zing finger rests in the major groove of the DNA and the side chains project outwards, contacting bases
- The identity of these side chains determines the specific DNA sequence that is recognised by each finger allowing sequence specificity of the protein
Why are multiple zinc fingers used in DNA binding?
Because one zinc finger only interacts with two nucleotide interactions - not strong so use multiple zinc fingers
Outline DNA gel electrophoresis
DNA migrate from minus to plus end of the field. If DNA was interacting with a protein then the DNA will be shifted several positions depending on which protein they interact with
What is the purpose of the salt gradient in DNA gel electrophoresis?
Using a salt gradient you can investigate the strength of protein interaction with DNA
The strongest interacting proteins will need high salt concentrations and weakly interacting will require low salt concentrations
What is the purpose of DNA foot printing?
To confirm DNA binding
How is DNA foot printing carried out?
Label DNA with radioactivity and then digest the DNA nucleotide by nucleotide
If a protein is interacting with the DNA sequence then you will see that you’re missing some of the degradation products - can see that protein is bound
What interactions occur involving a zinc finger?
Binds zinc to DNA
What interactions occur involving a leucine zipper?
Protein - DNA binding
What interactions occur involving an EF hand?
Binds calcium or magnesium in structural or signalling mode
What interactions occur involving a SH2 domain?
Binds phosphorylated proteins
What interactions occur involving a SH3 domain?
Binds proline rich motifs
What interactions occur involving a PH domain?
Binds phosphorylated lipids
What DNA binding domains are involved in the insulin signalling pathway?
- SH2 domain - binds phosphorylated tyrosine (Grb2 protein)
- SH3 domain - binds proline-rich motifs (Grb2 protein)
- PH domain - binds phosphoinositide lipids (IRS1 and Sos proteins)
Outline the insulin signalling pathway focusing on the protein domain interactions
- The activated receptor phosphorylates itself on tyrosine - This then recruits a protein called insulin receptor substrate-1 (IRS1) via a PTB domain of IRS1
- the PH domain of IRS1 also binds to phosphoinositides on the plasma membrane
- the activated receptor phosphorylates IRS1 on tyrosines, and one phosphotyrosine binds
the SH2 domain of the adaptor protein Grb2 - Grb2 uses two SH3 domains to bind to a proline-rich region of a protein called Sos,
- Sos also binds to phosphoinositides in the plasma membrane via its PH domain.
- Grb2 uses its other SH3 domain to bind to a proline-rich sequence in a scaffold protein.
- The scaffold protein binds several other signaling proteins, and relays the signal further
Outline the characteristics of the SH2 binding domain?
It is an important phospho-tryosine binding domain often involved in signalling mechanisms
- important in the formation of signalling complexes
- Binding occurs between the negative phosphate group and the positive amino acid
- Hydrogen bonds also contributes
Where is the original SH2 binding domain found?
Protein tyrosine kinase Src
Outline the characteristics of the SH3 binding domain
Use aromatic amino acid stacking (hydrophobic staking) to bind its ligand
- It is a polypro line binding domain
- Involved in linking signalling components and maintaining multi protein complexes
- The minimum consensus sequence for SH3 binding are proline - two amino acids - Proline
- Tyrosines and tryptophanes – are positioned such that they
can stack with the aromatic rings of proline residues in target proteins
Outline the characteristics of the PH binding domain?
- Involved in membrane binding, signalling and anchoring proteins to membranes
- The PH domain interacts with charged head groups of phospholipids anchoring the protein to the membrane
- Phospholipases and kinases use PH domain and have a direct role in lipid signalling.
How do we study protein protein interactions?
Biochemically
- Centrifugation, chromatography, pulldown reactions
Structurally
- X ray crystallography, NMR, electron microscopy
What is the role of a tag in affinity chromatography or immunoprecipitation?
Tag the protein of interest with a negatively charged flag peptide and have an antibody against the tag
- Can be used for rapid purification of tagged protein and many associated proteins
What is the immuno pull down of interacting proteins?
- Mix tagged protein with cell extract or other proteins to allow binding of complexes
- Add antibody to tag
- Add protein A coated beads
and centrifuge to recover complex. - WASH beads.
- Identify by mass spectrometry
or western immunoblotting. - Use a specific antibody targeted to the epitope tag attached to the protein of interest.
- The antibody protein complex is recovered using Protein A coated beads. Protein A is a bacterial protein that binds strongly to immunoglobulins.
- The Protein A beads are used to ‘immunoprecipitate’ the protein complex
What are the common tags used for affinity chromatography or pull down?
Glutathione-S-transferase (GST)
Hexa-histidine (6xHis)
What are the common tags used for immunoprecipitation?
HA peptide YPYDVPDYA
Myc peptide EQKLISEEDL
Flag peptide DYKDDDDK
What is GST affinity pull down?
- Recombinant DNA techniques are used to make fusion between a protein and glutathione S-trandferase (GST)
- Use fusion protein to ‘pull’ an interacting protein from a mixture, known as ‘pulldown’ in this case GST-pulldown.
- Resulting mixtures separated by SDS-PAGE and western blotted with antibodies for binding partners.
- If binding partners unknown, identify by mass spectrometry
What is yeast two-hybrid screening used for?
To identify interacting sequences
Outline yeast two hybrid screening
- Bait protein cDNA cloned into bait plasmid in the host yeast strain.
- A large library of random DNA prey plasmids are added
- Interaction between bait and prey after introduction into yeast allows DNA binding and activation domains to activate a reporter gene allowing it to survive
- Yeast colony survives on a restrictive growth media
- Correct Prey plasmid recovered and sequenced to find interacting protein.
What is the role of ELISA (enzyme linked immunosorbent assay)?
To measure the strength of protein interactions
Outline direct and indirect ELISA as a method of measuring the strength of protein interactions
Direct ELISA
- Antigen is attached to a 96-well plate
- Primary antibodies added to the plate. These antibodies have been tagged with a fluorophore or an enzyme, allowing the target protein to be identified
Indirect ELISA
- Secondary antibodies used to detect the primary antibodies by binding to the Fc domain
These antibodies have been tagged with a fluorophore or an enzyme
- Allows for amplification of the signal if protein is present in small amounts
What is fluorescence resonance energy transfer (FRET)?
It measures the strength of protein interactions
- The two proteins are labelled with blue fluorescent and green fluorescent protein
- If resonate green light then the proteins must be interacting with each other
- Because the green fluorescent protein absorbs blue light and admits green (have to be close enough to protein labelled with blue to absorb the light)
What is surface plasmon resonance?
Light source is projected onto a sensor chip and is reflected back to a detector
The light source causes surface plasmon resonance (electron resonance) which can form a trace
The binding of prey molecules to bait molecules on the sensor chip increases refractive index of the surface layer and therefore alters the resonance angle for plasmon induction. This shifts the position of the trace
The Kon (association rate) and the Koff (dissociation rate) of a given protein protein interaction can be read directly from these traces
Give the equation for the association rate of protein interactions at equilibrium
Kon[A][B] = Koff[AB]
[AB]/[A][B] = Kon/Koff = K = Equilibrium constant
Give an example of how protein protein interactions can be studied in vitro/
Vesicle movement along microtubules relies on kinesin interaction with microtubules and can be studied by light microscopy
- Can attach the head domain of the kinesin to glass plate to study how the microtubules move
How is DNA usually damaged?
Oxidation, hydrolysis or uncontrolled random
methylation of any of the four nucleotides
What can DNA damage lead to?
Change the bases coding properties, remove a base from the DNA and actually fragment the DNA by breaking the phophodiester backbone
What is the most common form of DNA damage?
Hydrolytic deprurination and deamination of bases
What occurs after the deamination of cytosine?
Become uracil
- Means that the base will be mispaired
- In DNA replication, one parental strand will therefore instruct DNA polymerase to pair it with adenine
What occurs after the depurination of adenine?
Adenine has been removed by hydrolysis
- Sugar and backbone still there but no base
- Meaning one parental trans in DNA replication will have lost adenine so there will be a full base pair deletion in that newly synthesised DNA sequence
How are deaminated and depurinated bases repaired?
Base Excision repair
Explain the base excision repair pathway
Where cytosine is deaminated to uracil, uracil DNA gycosyclase recognises this abnormally as the uracil in the original DNA strand is mispaired with a guanine which is recognised
- Uracil DNA glycosyclase then removes the uracil base
- Acts as a signal to activate two other enzymes
- The AP endonuclease removes the deoxyribose sugar and the phosphodiesterase removes the phosphate creating a single nucleotide gap
- DNA polymerase can then fill in the gap and use the guanine from the complementary DNA strand as a template to fill the gap with cytosine
- DNA ligase then seals the nick
What can ultraviolet radiation cause?
The formation of Pyrimidine Dimers, which can arrest DNA Replication or cause mis-reading of the DNA sequence by DNA Polymerase
Causing the formation of covalent binds between carbons in adjacent pyramiding rings - distorting the DNA and DNA polymerase ability to recognise the nucleotides
How is DNA damage by UV radiation fixed?
Nucleotide excision repair
- Excision nuclease is activated and creates two cleavages in the backbone, on either side of the damage
- DNA helicase then removes this fragment that he excision nuclease has created displacing several undamaged bases along with the damaged pair
- Recognised as a primer template junction (gap) for DNA polymerase and extends across the gap
- DNA ligase then seals the nick
What can defective nucleotide excision repair machinery lead to?
Makes skin vulnerable to UV light
People with these mutations have hypersensitivity to skin cancer
- Xeroderma pigmentosum - can be caused by mutations in the following genes - XPA, XPC, XPD, XPF, XPG
What are the homologues of E.coli Xeroderma pigmentosum proteins?
Uvr proteins:
UvrA, UvrB, UvrC, UvrD
Where does nucleotide excision repair usually occur?
It is tightly coupled to areas being transcribed
- Rapid response
- transcriptively active genes
- Enzymes involved (such as excision nuclease) are associated with RNA polymerase II
Why are double stranded DNA breaks dangerous?
Because it could lead to large fragments of chromosomes being lost
How are double stranded DNA breaks formed?
Ionising radiation
How are double stranded DNA breaks repaired?
- Non-homologous End Joining
2. Homologous Recombination (HR)
What is Non-homologous End Joining?
A rapid way of the body repairing double stranded DNA breaks
- rapidly joins together free ends of fragments that are in close proximity to each other
What are the risks associated with Non-homologous End Joining?
May join the wrong fragments together which can lead to loss of DNA fragments
What is homologous recombination?
The last line of defence in DNA repair
- It makes use of information that is in the undamaged homologous chromosome - high accuracy
Where is homologous recombination commonly needed?
In other forms of repair where there is a partial single stranded section of DNA - vulnerable to stress so may break completely
Outline the mechanism of homologous recombination
- Two sister chromatids (one damaged, one not) accurately align
- Exonuclease is activated and degrades the 5’ ends of the damaged chromatid to create two long 3’ overhangs
- RecA then promotes strand invasion of the undamaged template molecule by one strand from the damaged DNA molecule acting as a primer
- The complementary sequence to the 3’ overhang on the undamaged chromosome is displaced with the damaged one (forms a holiday junction)
- The 3’ end of the invading strand acts as a primer for DNA polymerase
- DNA polymerase extends the 3’ end cross the sequence that has been rendered single stranded on the other fragment - branch point mutation
- When it has all been copied, DNA helicase displaces the extended molecule and promotes its reanealing to the damaged DNA
- DNA polymerase synthesises across the gap on damaged chromatid using the newly synthesised extended molecule as a template
- DNA ligase seals the nick
What types of cancer can mutations in the components in homologous recombination lead to?
- BRCA2: breast, ovarian and prostate cancer
- ATM: Ataxia telangiectasia – leukaemia, lymphoma
- Fanconi Anaemia – complicated with leukaemia: 13 different FANC genes
How has knowledge about homologous recombination and cancer lead to cancer drug development?
Cells that are defective in this way are heavily dependant on other pathways for DNA repair
- Can therefore develop drugs that target other forms of DNA repair
- If these cells cannot use homologous recombination to other forms of DNA repair then those cells will die (synthetic lethality)- killing the cancer cells
What is a holiday junction?
The crossing over of one strand from one helix forming base pairs with the complementary strand of the other helix
How is homologous recombination used in meiotic cells?
Creates new combinations of alleles
- Create a double stranded break in one of the two chromosomes using the endonucleases: Spo11 (makes the initial cleavage) and Are11 (resects the 5’ ends, creating 3’ overhang)
- RecA is recruited and promotes formation of holiday junction (strand invasion)
- DNA polymerase fills I the gaps on both DNA strands using the DNA from the other chromatid as a template
- DNA ligase creates the second holiday junction
- The holiday junctions are then resolved and crossing over is complete
What is the main difference between homologous recombination in repair and in meiosis?
DNA polymerase uses strands from both chromatids as templates to fill in self created breaks in meiosis
What are the two possible ways that holiday junctions can be resolved in meiosis and which is used?
Pathway 1
- Internal strands of holiday junctions are broken and rejoined (same strands are broken and rejoined at each junction)
- This doesn’t achieve crossover as all four stands need to be broken - not used
Pathway 2
- At the holiday junction, the two outer strands are broke and rejoined but at the other holiday junction, the internal strands are broken
- The chromatids then rejoin with a successful crossover as each chromatid has a part of the other chromatid on it
How often does cell proliferation occur?
Typically one division every 24hrs in proliferating mammalian cells. Other cells do not divide once they are born and last for many years.
What are the four stages of the cell cycle?
G1 - Gap 1
S - DNA replication
G2 - Gap 2
M.- Nucleair division
What stages of the cell cycle make up interphase?
G1, S, G2
What occurs during M phase of the cell cycle?
Prophase – condensation of sister chromatids (identical copies).
Metaphase – attachment of the mitotic spindle to the kinetochore by microtubules.
Anaphase – separation of sister chromatids.
Telophase - Nuclear membrane reforms
What are the types of cell division in yeast?
Fission and budding
What are the advantages of using yeast as a genetic model for cell cycle?
Rapid division rate <1hr
Cell cycle control genes are highly conserved
Yeast can be grown as haploids or diploids
What genetic tricks can we use to allow for identification and investigation of potentially lethal mutations?
- Diploids can be used to maintain lethal mutations (they won’t die as there is a second wild type gene) and then they can be studied as haploids
- Temperature sensitive mutations allow growth at a permissive temperature - vary the temperature depending at what stage in the cell cycle you wish to investigate
What are Cdc genes?
Cell division cycle genes
What are the advantages of using Xenopus leaves as a biochemical model for cell cycle?
Easy to collect eggs
Rapid division rate
Large size makes purification of proteins easier
Can be manipulated by injection of RNAs or chemical into the oocyte
What is meant by cell free mitosis and what are its uses?
Cytoplasm from frogs egg is added to the nuclei from a frogs sperm and ATP is added
Mitosis
One can deplete the cytoplasm of different proteins using antibodies
One can remove cytoplasm at different stages to study changes
(eg in protein phosphorylation) over time.
When are the checkpoints in the cell cycle?
Start checkpoint
- End of G1
- Is the environment favourable for cell division?
G2/M checkpoint
- As enters mitosis
- Is all the DNA replicated?
- Is the environment favourable for cell division?
Metaphase to anaphase transition
- trigger anaphase and cytokinesis
- Are all chromosomes attached to the spindle?
What are cyclins?
- Cyclins are proteins that are expressed at different levels during the cell cycle
- When present, cyclins bind to specific kinases (called cyclin dependent kinases, Cdks) to activate them.
- CDKs phosphorylate many proteins that are specific to certain stages of the cell cycle.
What other proteins also modify CdK activity?
Wee1 kinase phosphorylates Cdks to inactive them
Cdc25 is a phosphatase that removes the phosphate group and activates Cdks
What are APC’s (anaphase-promoting complex)?
A ubiquitin ligase
It ubiquinates M-cyclin, S-cyclin and securin
Involves E1 and E2 enzymes which cause ubiquitination and therefore degradation of the cyclin in the proteasome
What is a securin?
A protein that holds together sister chromatids
What is meiosis?
- Diploid organisms have two versions of each chromosome (homologues).
- Homologues are either paternal or maternal.
- Only one homologue for each chromosome is packaged into a gamete.
- Meiosis resembles mitosis except that there are extra steps that segregate homologous chromosomes.
- Pairing of homologues before segregation allows for crossing-over (homologous recombination)
What occurs in meiosis I?
Crossing over and segregation
- DNA replication
- Homologue pairs line up on spindle and are pulled apart
What occurs in meiosis II?
Resembles mitosis
Main difference to mitosis is that cells in meiosis II are haploid not diploid
What occurs in meiotic prophase I?
Homologues pair up
- Pairing in facilitated by the synaptonemal complex (proteins) as well as DNA base pairing between homologues.
- Homologous recombination between nonsister chromatids serves two purposes:
1) It aligns the chromosomes up ready for anaphase and facilitates formation of the synaptonemal complex.
2) It allows for genetic recombination between paternal and maternal DNA on the same chromosome
Why is genetic variation important?
For evolution
What are the problems associated with meiosis 1?
Mistakes during meiosis I result in gametes with an extra chromosome or lacking a homologue.
This is called nondisjunction and the cells that arise from these gametes are called aneuploid.
4% of mammalian sperm is aneuploid, 20% of mammalian eggs are aneuploid.
What is the normal relationship between the level of transcription and level of expression?
Usually increased transcription means increased expression
What can occur that stops increased transcription meaning increased expression?
RNA may be transcribed but not translated into a protein
Protein may be readily degraded
What kind of interactions are essential in the regulation of transcription?
DNA protein interactions are essential for regulation of transcription
How do DNA binding proteins interact with DNA?
DNA binding proteins are positively charged which allows the protein to stick to the negatively charged backbone
- The charge allows the protein to be in close proximity to the DNA molecule but the alpha helicies reach into the major groove to interact with specific bases
What amino acids are responsible for DNA binding proteins having a positive charge?
Lysine or Arginine
How do DNA binding proteins interact with specific bases?
Reaches into the major groove of the DNA and forms two hydrogen bonds between asparagine and adenosine
What is Rox1?
A DNA bindin protein that has 8 binding sites in yeast
- 3 of the binding sites are in the HEM1 3 gene
- 4 are in ANB1 gene
- 1 in ROX1 gene itself
Why do some DNA binding proteins have multiple binding sites?
The binding sites are not all identical
- This could be because it can change the strength of the binding of the proteins (changes the affinity of the binding sites) facilitating regulation
How can multiple binding sites be represented?
By the frequency of each bases at each position
A consensus sequence (average)
Sequence logos
What is a motif?
A protein structure that serves a purpose?
Name some DNA binding motifs?
Helix turn helix
Zinc finger
Leucine zipper
Helix loop helix
Give the characteristics of the helix-turn-helix DNA binding motif
It is made up of two alpha helices and one enters the major groove and the other sites on the backbone
- Binds as dimers
- Two recognition sequences in major groove
- The proteins bind head to head
- The binding site is palindromic
Give the characteristics of the zinc finger DNA binding motif
Uses a zinc atom, interacts with 4 amino acids
Allows it to position the alpha helix in the major groove
Interacts with Arginine and histamine
Proteins with zinc fingers usually have more than one
Give the characteristics of the leucine zipper DNA binding motif
two alpha helical monomers for a dimer held together by hydrophobic amino acids (e.g.. leucine)
The two alpha helices enter the major groove
Can interact as homodimers (2 copies of same recognition sequences) or heterodimers (2 different sequences)
Give the characteristics of the helix loop helix DNA binding motif
Related to leucine zipper but has loops which enables more flexibility
What is the role of DNA binding motifs?
The cooperatively of DNA binding motifs increases binding strength
What are the two techniques of identification of DNA binding proteins?
DNAse I footprinting
EMSA - electrophoretic mobility shift assay
Outline DNAse I footprinting
Radioactively label of one end of the DNA (32P)
Mix with cell extract (or purified protein)
Add DNAse to partially digest the DNA
The DNA binding protein protects the bound DNA from cleavage - able to identify where the protein is
Heat sample to destroy the DNAse and release the binding proteins.
Run samples by gel electrophoresis
Outline the process of EMSA
Radioactively label of one end of the DNA
Mix with cell extract (or purified protein)
Run samples by gel electrophoresis
Shifts to a slower rate of movement through the gel id a DA binding protein is bound
What are the types of transcription factors?
Permissive - General transcription factors which are necessary for all transcription and bind to the promotor (not regulatory)
Specific or regulatory - Activators (increase transcription of neighbouring genes) and repressors (reduce transcription of neighbouring genes)
Why is DNA looping important in transcription?
Plays an important role in forming DNA/protein competes
Allows DNA to loop around and interact with binding sites on proteins
Chromatin doesn’t bend easily so binding sites aren’t more than 500 base pairs away
What is an enhancer?
A binding site for transcriptional activator
Are promiscuous - activate any genes in that area
What is a silencer?
Binding site for transcriptional repressor
What is the role of insulator elements?
Prevent the enhancer for activating all of the genes
What is a genetic switch?
An input that alters gene expression
- Each switch is responding to extrinsic or intrinsic regulation
What are the ways to regulate a transcription factor?
Protein synthesis
Ligand binding
Protein phosphorylation
Addition of second subunit
How do transcription factors interacts synergistically with each other?
Having 2 binding sites can increase transcription:
- They may bind to each other and stop it from falling off the DNA - would need to break two interactions to fall off not just one
- Also may work by binding one of the transcription factors to the DNA and causing it to unwind from the nucleosome making another binding site accessible
What is the difference between genetic alternations and epigenetic alternations?
Genetic alterations to DNA sequence can permanently affect gene expression
whereas epigenetic changes to chromatin structure can also modulate gene
expression but they do not alter DNA sequence and are reversible
Where are epigenetic changes reversible?
In the germ line
- May persist for the entire life of a somatic cell but can be erased in germ line
What is meant by epigenetic landscape?
Different cell fates during development are the end of distinct journeys through an epigenetic landscape
- Encounter different signals
What part of the chromosome is covalently modified?
The N-terminal lysine-rich tails of core histones project radially from the nucleosomal core and are covalently modified
What are the most common covalent modifications?
Acetylation of lysines in core histone N terminal tails
Methylation of lysine and arginines in core histones N terminal tails
- Methylation can be mono, di or try methylated
What is the role of histone acetyltransferases (HATs)?
Responsible for acetylation of lysine resides in core histones
What is the role of histone methyltransferases (HMTs)?
Responsible for methylation of lysine and arginine
What is the difference between histone methyltransferases (HMTs) and histone acetyltransferases (HATs)?
Histone acetyltransferases - can modify many different lysine residues in core histones Histone methyltransferases (HMTs) - highly specific so requires specific HMTs for specific lysine residues
What is the role of histone demethylases and histone deacetylases?
Remove the acetylation and methylation of lysine residues
What is the effect of different lysine residues being methylated?
Depending on which residue is methylated, methylation can either promote transcription activation or repression
- Lysine 4 methylation causes transcriptional activation
- Lysine 9 or 27 methylation causes transcriptional repression
What is the effect of acetylation of lysine residues?
Exclusively activates transcription on whatever lysine it occurs
Is acetylation or methylation the dominant modification?
Acetylation
- Can exclude the action of methylation on the same lysine that would have lead to transcriptional repression
How do histone acetyltransferases (HATs) lead to transcriptional activation?
Acetylation of histones creates binding sites for transcriptional activation factors that contain a bromodomain
The bromodomains then bind to the acetylation and promotes transcriptional activation
Give some evidence for histone acetylation occurring mainly at promotor sequences?
In yeast
- Analysis measured the acetylation of the histones at the promotor sequence of each gene in a section
- Can see that acetylated histones increases at each promotor sequence
How do histone methyltransferases (HMTs) lead to transcriptional repression?
On lysine residues 27 and 9 on histone 3
- Create binding sites that contain chromodomains
- These chromodomains bind and cause transcriptional repression
What is meant by chromodomains being a specific code reader?
They only bind to methylations on specific lysine residues - 9 and 27
How do histone methyltransferases (HMTs) lead to transcriptional activation?
On lysine residues 4 on histone 3
- Creates binding sites for transcriptional activators that contain a PHD zinc finger domain
- This domain binds and either sustains or increases transcription
What is the role of the epigenetic code?
An epigenetic code that lies on top of the genetic code and governs when and where genetic information is expressed
How do transcription activator proteins work in chromatin?
- Interactions between chromatin remodelling factors and transcriptional activators that can change the density of nucleosomes in a specific region - can male recognition sites for other activation proteins available
- Selective histone removal
- Recruit histone acetyltransferases - can recruit the code writers and reader
How do transcription repressor proteins work in chromatin?
- Preventing activators binding to promotors - competitive DNA binding
- Masking the activation surface
- Direct interaction with the general transcription factors
- Recruitment of chromatin remodelling complexes
- Recruitment of histone deacetylases
- Recruitment of histone methyl transferase that causes transcriptional repression
What is the polycomb group of proteins?
The histone code writing and reading system
- made of two complexes
- One that encodes a set of histone code writers
- One that encodes a set of histone code readers
Give the characteristics of the code writing component of the polycomb group of proteins
Polycomb repressive complex 2
- Contains a histone methyl transferase enzyme called enhancer of Zeste (EZH2) at its core
- Writes into chromosome on histone H3 lysine 27
Give the characteristics of the code reading component of the polycomb group of proteins
Polycomb repressive complex 1
- Contains a chromodomain containing protein (called polycomb) at its core
- Recognises lysine 27 on histone H3
What disease has been associated to over expression of EZH2?
Abundant in many cancers
The methylation of H3K27 (Histone H3 lysine 27) is over expressed
An inhibitor of EZH2 blocks the enzyme and also kills the mutant lymphoma cells
What is the role of DNA methyl transferases (DNMTs)?
Addition of methyl groups to cytosine residues
What is the relationship between histone methyl transferases and and DNA methyl transferases?
They physically interact with each tiger and mutually reinforce each other effects
What is mammalian X-chromosome inactivation?
Selective innovation of x chromosome in females during development
- Silencing is random and is the propagated clonally - all the progeny of each cell in which the silencing design was taken inherit the same silenced x chromosome
Give an example of mammalian X-chromosome Inactivation and explain why it occurs?
Calico cat
- Only female
- A pigment mutant that has patches of orange and black fur
- Because its heterozygous on x chromosome (males only have one allele so is all orange/all black)
- The patches occur because during development random inactivation of X chromosome occurred. So in black patches of fur the orange allele was silenced and in orange patches the black allele was silenced
How is mammalian X-chromosome Inactivation initiated?
Production of RNA molecule (Xist RNA) form inactivation centre (XIC) from the x chromosome that is to be inactivated
- Its function is not to encode but to decorate the chromosome from which it was transcribed and to stimulate its own synthesis
- Recruits polycomb proteins causing repression pf the locus
- Causes condensation of the X chromosome inactivating it
Outline the history of the codon
1944 - Avery provides evidence that DNA carries genetic information
1953 - Watson, Crick, Franklin - DNA structure
1966 - Nirenberg, Ochoa, and Khorana elucidate the genetic code.
Define codon
Three bases that encode an amino acid
What is meant by the genetic code being degenerative?
Amino acids are specified by more than one amino acid (61 codons, 20 amino acids)
What is a reading frame?
Where you start reading the codon from
- Three possible ones
How is the correct reading frame ensured?
By a start codon - where translation always starts
AUG (methionine)
What are the stop codons?
UAA, UAG, UGA
What is the structure of tRNA?
One end base pairs with the codon - the anticodon loop
The other end carries the amino acid at the 3’ end
Intermolecular base pairing within the tRNA gives it structure
Some of the nucleotides in tRNAs
What is the ratio of tRNA molecules to codons?
Not 1:1
- eg. there are 6 codons for serine but only 3 tRNAs
Why is the ration between tRNA molecules and codons not 1:1?
To allow for wobble base pairing
- The third base (wobble base) cn pair with one of two different bases - provides leniency and allows tRNA anticodon to pair with more than one codon
How many tRNA molecules do bacteria have?
31 tRNAs for 61 codons
How is the tRNA molecule attached to the amino acid?
- tRNA is charged by the addition of AMP
- The enzyme (aminoacyl-tRNA synthase) causes the hydrolysis of ATP and adds the AMP to C terminus of the amino acid. The phosphate carries high energy
- The same enzyme binds to tRNA and on 3’ end is joined to amino acid through the remaining O
- Releases energy, forming ester link between tRNA and amino acid
- Energy from the ATP hydrolysis remains in the ester linage so is known as charged tRNA
How is it ensured that the correct tRNA molecule is attached to the correct amino acid?
Requires two adapters:
- The synthase that pairs the correct amino acid to the correct tRNA is specific - they are specific to individual tRNAs. Amino acids have to fit into two different pockets in the synthesise (before and after AMP addition). The correct amino acid will have3 high affinity or the active sit pocket of its correct synthatase so is favoured. Some amino acids have similar structures so are removed in the second pocket of the syncretise by hydrolysing from the ADP
- The tRNA that pairs the correct codon to the correct amino acid within the ribosome. Ensures correct codon through complementary base pairing
How does elongation of an amino acid sequence occur?
Using enzyme peptide transferase
- High energy ester linking between amino acid and tRNA
- N terminus of aminoacyl tRNA breaks this high energy ester bond of the adjacent amino acid and tRNA, removing that tRNA and forming a low energy covalent bond to the amino acid
Where does translation occur?
The ribosome
Give the structure of the ribosome?
- It is divided into two parts (subunits): The large subunit catalyzes polymerization while the small subunit facilitates the tRNA/mRNA interaction.
- The subunits come together on the 5’ end of the mRNA, process along the mRNA at two amino acids/second, then separate at the stop codon.
How does translation occur in the ribosome?
Charged tRNA enters the ribosome at the A-site
Petidyl transferase catalyses amino acid addition, conformational changes move the tRNAs to the E- and P-sites.
Conformational changes move the small subunit three nucleotides.
tRNA leaves the E-site
What is the purpose of elongation factors and give an example of one?
Help translation and improve accuracy (EF-1)
How do elongation factors work?
Once the anticodon is bound, EF-1 causes two delays before the petidyl transferase can act: first it must hydrolyse GTP to GDP. Next it has to dissociate from the tRNA. Checkpoint.
- Both of these lags allow time for incorrectly bound tRNAs to fall off. Some of the correct tRNAs also fall off, but at a slower rate.
- The hydrolysis of GTP occurs more rapidly if the codon and anticodon are correctly matched.
- If synthesis is made in the absence of EF-1 then there are more errors in the protein sequence.
Give the characteristics of peptidyl transferase?
It is an RNA not a protein
What is a ribozyme?
A non protein based enzyme
- The ribosome is a ribozyme
Explain how the ribosome is a ribozyme?
Large subunit rRNAs form a massive structure that contains most of the catalytic activity including that of the petidyl transferase.
How is it only methionine that can start translation?
Only the methionine tRNA with elf-2 (initiation factor) can bind to the small subunit of the ribosome alone - the ribosome recognises the 5’ cap and associated initiation factors
How does eIF-4E work?
It is a eukaryote initiation factor
- mRNA that has a cap and tail which are bound by eIF-4G and eIF-4E to form a loop. This is a checkpoint for broken mRNA as broken DNA will not have these initiation factors
Can more than one ribosome bind one mRNA?
Yes multiple ribosomes can bind
- ribosomes are spaced about 80 nucleotides apart on a polysome
How do stop codons work?
Stop codons are recognized by release factors. These look like charged tRNAs (molecular mimicry), and enter the A-site. This results in dissociation of the ribosome.
When does the folding of a protein occur?
Folding of the protein begins immediately after leaving the ribosome
What is a molten globule?
Many proteins initially fold into roughly the correct confirmation. This is called a molten globule
What are the first protein foldings that occur usually?
As the protein exits it folds rapidly putting hydrophobic side chains in the middle to achieve a lower energy state.
What occurs when a protein is misfolded?
An incorrect step may reduce the energy state but blocks further folding. This leads to a dead end.
Misfolded proteins generally have exposed hydrophobic regions that can lead to aggregation.
How can protein misfolding be saved?
By molecular chaperones
- Reverse incorrect steps
What are the two major classes of molecular chaperones and how do they work?
The two major classes are hsp60 and hsp70
- They are named heat shock proteins because their expression is elevated when the temperature is raised above normal
- High temperatures cause properly folded proteins to unfold - so chaperones function during normal folding as well as when a cell has been overheated
How do hsp70 molecular chaperones work?
The hsp70 class work directly on proteins as they exit the ribosome, binding to exposed hydrophobic amino acids
How do hsp60 molecular chaperones work?
They put misfolded proteins into isolation
- The hydrophobic entrance binds to the protein partially unfolding it
- The GroES cap then seals the protein inside for 15 seconds allowing refolding
How many newly synthesised proteins are immediately recycled?
1/3 - due to misfolded
- Are therefore polyubiquitinated and targeted for destruction in the proteasome
What diseases can be caused by protein aggregates?
CJD, Huntington’s disease and Alzheimer’s disease are all associated with large, extracellular protein aggregates.
What is a protein aggregate?
Protein aggregates are large and protease resistant (difficult for the cell to get rid of) and they can lead to the cell death. They sometimes cause a chain reaction to misfold more proteins
Define transcription
Transcription is the process of RNA synthesis from a DNA template.
What are the three main types of RNA?
mRNA - messenger RNA, codes for proteins. - 3-5% total RNA tRNA - transfer RNA, participates in translation. - - There are 49 families of tRNAs. Each carries an aminoacid and has a specific anticodon loop. rRNA - ribosomal RNA, major constituent of ribosomes. - There are 4 main rRNAs
What is the difference between RNA and DNA structure?
- Contains ribose (instead of deoxy-ribose)
- Contains Uracil instead of Thymine
- Synthesised as a single strand
- RNA is very unstable
What kind of base pairing does occurs in RNA?
Watson Crick paring
- A=U,G=C
Can also have non Watson Crick pairing due to wobble base pairing so in the wobble position (3rd) G could pair with C or U
What is the secondary structure of RNA?
Stem-loop structure
- Formed due to base pairing
What is the tertiary structure of RNA?
When the secondary structure folds into a there dimensional structure
What is the function of RNA polymerases?
Perform RNA synthesis
What are the types of RNA polymerases?
RNA polymerase I - ribosomal RNA RNA polymerase II - protein-encoding genes RNA polymerase III - tRNA, small nuclear RNA, and 5S rRNA
What are the differences between RNA and DNA replication?
Multiple RNA Polymerase bind on the same gene
No primer needed
Only one strand of DNA is used as the template
Transcript does not remain bound to template
Higher error rate
Which strand of DNA does RNA polymerase copy?
Can use either strand of DNA as the template
The RNA synthesised will therefore be a copy of the other strand and the template strand will be the antisense strand
What is meant by supercoiling?
Chromosomes are too large to spin during replication so become knotted
How is supercoiling overcome?
Topoisomerases release supercoils to allow progression
How doe RNA polymerase know where to begin on DNA?
Has specific promotor sequences that act as binding sites
- eg. TATA
What RNA processing occurs after its synthesis?
- Splicing of introns: eliminates non coding regions of mRNAs to generate mature mRNA for protein synthesis
- Capping of 5’ end: necessary for stability, binding of mRNA to ribosomes and initiation of translation.
- Polyadenylation of 3’ end: part of the termination process (addition of poly-A tail). Determine mRNA stability, helps mRNA nuclear export and translation.
What is meant by splicing?
Removal of introns and joining of exons
Specific to eukaryotes transcription.
Introns-Exons present in the DNA and pre-mRNA, not in mRNA
Some mRNA can be spliced in more than one way - alternative splicing
How does splicing occur?
- 2’ OH of branch site A attacks phosphodiester bond on donor site G.
- Cleavage at the donor site. Formation of lariat.
- 3’ OH of donor site G attacks phosphodiester bond on acceptor site G freeing the lariat.
- Lariat is degraded.
What is responsible for mRNA splicing?
The spliceosome
What is the spliceosome?
A nuclear complex made of about 150 proteins + 5 RNAs
Responsible for mRNA splicing
What are the functions of the spliceosome?
- Recognize 5’donor and branch sites
- Bring sites together
- Catalyzes RNA cleavage
How does synthesis of the 5’ cap occur in mRNA?
- Occurs in all eukaryotic mRNAs
- Unusual because its a 5’ to 5’ linkage so requires a specific enzyme
- Cap added when mRNA is 20-40 nucleotides long
- Cap is made up of methylated guanine
How does polyadenylation occur?
- Requires enzymes cstF (cleave stimulation factor) and cpsf (cleavage and polyadenylation specific factor). These enzymes move with RNA polymerase and then move to the 3’ end of mRNA and cleave it
- Poly A binds and 200 A nucleotides
- Poly A binding proteins assemble and determine the final length
- Important to initiate termination of RNA transcription
- Proteins are held in place by CTD (pol II c-terminal domain) - highly phosphorylated
What is the function of restriction enzymes?
They cut DNA into manageable sizes - restriction fractions
How do restriction enzymes work?
- They act as dimers and recognize short palindromic DNA sequence
- They have precise recognition sequences
- Some leave overhangs, others cut the DNA flush (in the middle) and are called blunt restriction enzymes
How can restriction fragments be separated?
Using gel electrophoresis
Outline the process of gel electrophoresis?
A current is applied
- DNA fragments migrate towards the anode because DNA is negatively charged
- The gel retards the migration of DNA fragments, separated the fragments by size - smaller fragments migrate faster because can fit through gaps in the gel
- Froms DNA bands
- Use a razor blade to chop out arose with DNA band to purify each one separately
What gel is used in gel electrophoresis?
Agarose gel
- Has a well (dip) in it where the DNA is added
Why is ethidium bromide dye requires in gel electrophoresis?
Because DNA in solution is transparent so requires a dye that will fluoresce when it is bound to DNA
What is the role of the enzyme ligase?
Joins DNA to create recombinant DNA
How do cohesive termini (sticky ends) ligate?
They can ligate because they can hybridise - the sticky ends are complementary to each other
Give an example of two restriction enzymes with different recognition sites that can produce sticky ends with the capability of hybridising with each other?
SpeI and XbaI
- Their overhangs are cohesive
What is a plasmid?
Plasmids are small, circular, extra-chromosomal DNA that occur naturally in bacteria.
What are the useful properties of plasmids?
They have their own origin of replication that usually results in about 50 copies of the plasmid being made in each bacteria
They usually carry antibiotic resistance genes
What is a plasmid vector?
Plasmid vectors are made from plasmids, usually by adding a bunch of restriction enzyme sites in one part of the plasmid (called a multiple cloning site)
How many bases of DNA can a plasmid vector hold?
<30 kilo bases of DNA
How many bases of DNA can a Bacterial artificial chromosome hold?
<300 kilobases
How many bases of DNA can a yeast artificial chromosome hold?
< 3 megabases
What is meant by transformation in DNA cloning?
Putting the DNA into the bacteria
What are competent bacteria?
Competent bacteria are bacteria that are ready to take up new DNA
How does transformation occur?
Transformation involves mixing bacteria with the plasmid DNA and creating temporary holes in the cell membrane (electroporation or chemical treatment)
This process is not very efficient, so the treated bacteria are selected on antibiotic plates
Once DNA has been implanted into the DNA, how are does cloning occur?
Colonies grow that contain thousands of bacteria, that each contain 50 plasmids that all originated from a single plasmid
Single colonies are lifted from the plate to start a liquid culture, the plasmid can be easily purified from the bacteria and stored or analysed
Where do you get DNA to start with? (for cloning and analysis)
Making a library of genomic clones or CDNA
What is a library of genomic clones?
Contains all the regulatory sequences as well as just coding DNA
- Allows the study of transcriptional information
What is a library of cDNA?
Only contains genes that are expressed as cDNA is made from RNA
How is a cDNA library made?
- Extract and purify mRNA
- This isn’t very stable so it is converted to DNA by reverse transcriptase enzyme (only found in retroviruses)
- It makes dsDNA from ssRNA
- Ligate into bacteria and culture and purify the clone - ensure only one insert into each plasmid
- Each bacteria only uptakes one plasmid
- Each bacteria only forms one colony
- So each colony expands to one single mRNA which can then be amplified
What is a transcriptome?
Only the genes that are expressed in the original tissue (as opposed to genome which is all genes)
What is the problem with trying to find desired gene from clones?
Many of the clones will be house keeping genes
Trying to find desired genes (e.g.. regulatory genes) will be quite rare
Genomic labs often do this
How is a genomic library formed?
Purify out genomic DNA and make restriction digest
Represents the entire genome
How does dideoxycytidine terminator sequencing occur?
- Start with a piece of unknown DNA (template)
- Heat and denature (100 degrees)
- Add primer which will start DNA polymerase reaction
- Allow to cool with primer that binds to specific part of the sequence
- Add DNA polymerase to dNTPs (nucleotide bases)
- DNA polymerase will bind to primer and add nucleotides
- When DNA polymerases adds a ddNTP (a deoxy terminator) the strand cannot be extended
- Add a small amount of fluorescently labelled DDNTIPs (ddGTP, ddATP, ddTTP, ddCTP)
- This wil cause the different chains to be terminated at different times
- Can use gel electrophoresis to see which position each one is terminated at and use different coloured fluorescent dye to identify which base it was
Why does the addition of a deoxy terminator (ddNTP) result in termination of the chain?
Because it is missing an OH at the 3’ end meaning another base cannot be added
How is each ddNTP identified in dideoxycytidine terminator sequencing?
While the sequencing gel is running, an electronic camera takes pictures of bands and measures intensity - can read 1000s of nucleotides
What kind of sequencing is used if the DNA is large than 1kb?
Progressive sequencing or shotgun sequencing
What is progressive sequencing?
Used when DNA is larger than 1kb
- Ends of clones are sequenced using primers from the vector
- Primers are designed based upon the new sequence and another round of sequencing is performed, and so on until sequences meet in the middle
- Uses large genomic clones
What is shotgun sequencing?
Make a genomic plasmid library and sequence the ends from each clone using primers from the vector
The short sequences are assembled into a contig by a computer program
The overlaps are then studied
What are the advantages and disadvantages of shotgun sequencing?
Advantage
- Requires no thought (eg primer design) -> automated
Disadvantage
- Need to sequence more than 6X the size of the genome to get large contigs (inefficient)
- Will always be gaps
What is the size of the human genome?
- 2kb
- therefore cannot use dideoxycytidine terminator sequencing
How do we find genes in the nucleotide sequence?
Use gene prediction software
- prediction often involves scanning the sequence for promoters, start & stop sequences and intron splice sites
Use your computer to translate the DNA in all 6 reading frames
- Then search for similarities to known proteins (BLAST)
What is blast protein alignment?
Input the amino acid sequence of a proposed protein
The Blast program searches huge databases for other proteins which have similar sequences
Then shows the alignment of the protein to a known protein
Similarity found between protein sequences suggests that the proteins evolved from the same common ancestor and that the proteins have similar molecular functions
What are microarrays used for?
Microarrays allow us to compare the transcriptomes of different tissues to each other, eg normal liver tissue to cancerous liver tissue
What is meant by microarrays being a high throughput method?
It is small scale, fast and automated
How do microarrays work?
A microarray is a large grid with lots of segments - very small - on glass slides manufactured by robots
- Each position in the grid contains one cDNA (as the antisense strand) - one spot for every gene in the genome
- Purify mRNA and tag with a fluorescent dye
- If the mRNA matches any cDNA on the array it will hybridise and will see the fluorescent dye, after rinsing off unbound mRNA
- Use specialised camera to measure the intensity of fluorescence
How can microarrays be used to investigate genes involved in certain cancers?
Compare the fluorescence of the microarrays of normal tissue and tumour tissue (of a specific region)
- If there are genes that are present in normal tissue but not in the tumour then these are potential tumour suppressor genes
- If there are genes that are present in the tumour and not the normal tissue then these are potential oncogenes
What are the three different ways to identify genes?
- Making a library of cDNA clones
- Making a library of genomic clones
- Microarrays
How can you analyse genes?
Mouse genetics
- Gene replacement
- Gene knock out
How is gene replacement a way of analysing genes?
Gene Replacement usually makes a small changes to the endogenous gene.
For example if you find a mutation present in human patients, you could test whether that mutation causes the disease symptoms in mouse by making the same change in the corresponding mouse gene.
How is gene knock out a way of analysing genes?
This completely removes the gene to determine its function.
To begin a knock-out project, you must first have a genomic clone of your gene.
How do we knockout a single gene in mice?
- First acquire a genomic clone of the gene and insert NEO directly into an exon (destroying the activity of the gene). TK is placed off to one side
- The sequences from the target gene are called homologous arms because they are the only sequence with homology to the mouse genome
- Then we introduce the construct into mouse embryonic stem (ES) cells using cell culture techniques.
- Next the cell’s DNA repair machinery recombines the construct into the mouse genome.This is not very efficient!
- Homologous recombination occurs sometimes (knock-out). When it does occur, the TK gene is lost.
What is the function of double selection in gene analysis?
To identify the knock out
How is double selection used?
We then use the selectable markers (Neo and TK) to identify colonies that are the result of homologous recombination
Cells that have integrated the Neo gene are now able to grow in Neomycin containing media.
Cells that have integrated the TK gene along with Neo will die when grown in GANC media
How do we ensure that the gene is knocked out in all tissues?
The selected cell line is then reintroduced into mice embryos.
The first generation are mosaic (a mixture of cells from the stem cell line and the mother). Their gonads are also mosaic.
These mosaic animals are bred to generate non- mosaic carriers of the transgene (2nd generation).
The carriers are then interbred to create homozygous mutant animals (3rd generation)
Give an overview of forward genetics
1) randomly mutate the genome
2) look for interesting phenotypes in the offspring
3) identify the gene that causes the defect
Why do we perform forward genetics in mainly yeast, C.elegans, Drosophila and zebrafish?
Because random mutagenesis affects the whole genome, so we have to analyze many mutagenized animals to find interesting phenotypes
These animals are cheap and have a short generation time
What type of mutations are forward genetic screens usually used for?
Recessive
What is the difference between forward and reverse genetics?
In forward genetics we start with a phenotype to find the gene
In reverse genetics we know the gene and we want the function
What technique is used in forward genetics?
Positional cloning (linkage analysis) - A way of using a genetic map to find the mutation and identify the gene
How are forward genetic screens dome in flies?
- Mutagenize male fly so that each sperm has a different set of mutations
- Cross with a healthy female to make viable offspring
- Outcross male heterozygous off spring with wild type females
- In cross family to see homozygous embryos - 1/4 offspring will show a phenotype
- Requires 3 generations
How can you tell if different mutations that lead to the same phenotype are just alleles of the same gene?
Take carriers (heterozygous) of the to mutant and cross them together
- If alleles of the same gene then 1/4 of the offspring will show the phenotype - mutations fail to complement each other
- If not the same gene then no offspring will show a phenotype - mutations complement each other and therefore are mutations of different genes
What is complementation analysis?
A technique that allows mutations to be sorted into distinct groups that correspond to each individual genes
What is the purpose of linkage analysis?
Used to identify genes
How is linage analysis carried out?
By analysing recombination between our allele and a known marker on the same chromosome, we can determine whether the gene and marker are linked.
The greater the distance between the gene and the marker, the more frequent crossing-over occurs during meiosis
- Can then calculate recombination frequency
How do you calculate genetic distance?
(Number of recombinant gametes/total number of gametes) x100
How is linkage analysis used to analyse genes involved in human disease?
Single Nucleotide Polymorphisms (SNPs) are markers that easy to analyse and vary from individual to individual. Over 1 million SNPs have been placed on the human genome (ie we know where they are)
DNA samples are taken from the mother and her children and hundreds of SNPs are tested. If a SNP is always present in diseased children, but not in healthy ones, then we know the gene is linked to the SNP.
How do mutations affect gene function?
Changes in regulatory sequences
- in the DNA that affects transcription. (Regulatory sequences such as enhancers)
Changes in non-coding sequence of the transcript
- may affect RNA splicing, stability or translation.
Changes in the coding sequence
- may alter an important amino acid affecting folding of the protein may create a premature stop codon -> truncated protein.
What is amorphic mutation?
A non-functioning - missense mutation that completely inactivates the DNA binding domain
- in the heterozygous there is normally enough gene product from the one wild-type copy. Haplosufficient.
- in homozygous there is a strong phenotype due to no transcriptional activation. This is recessive.
What is a hypomorphic mutation?
A weakened - missense mutation that weakens the DNA binding domain
- In the heterozygous there is normally enough gene product from the one wild-type copy. The mutant form may also dimerize with wild type and still activate transcription.
- In the homozygous there is a mild phenotype due to poor transcriptional activation. Complex forms on DNA but is often falling off. This is recessive.
What is an antimorphic mutation?
A dominant - missense mutation that destroys the dimerisation domain
- In the heterozygous, the mutant form binds DNA but does not dimerise with the wild type and thus does not go through a conformational change to become active.
Transcription is compromised and is only activated when two WT proteins land together. This is dominant.
- In homozygous it is completely inactive
What is a hypermorphic mutation?
An overactive - missense mutation that results in activation that is independent of dimerization
- In the heterozygous, the mutant form binds DNA is active all the time. We call this constitutively active. This increases the overall activation of transcription. This is dominant.
- In the homozygous - it is the same
What is an isoform?
Different forms of proteins that can be formed by exactly the same genes
How can an isoform by transcribed?
Alternative splicing
- Optional exon
- Optional intron
- Mutually exclusive exons
- Internal splice site
How many of drosophila and human genes are alternatively spliced?
40% of drosophila genes and 75% of human genes
How is it ensured that the reading frame is maintained when alternative splicing occurs?
The sequence added/taken away is divisible by three
Give an example of a drosophila gene that uses alternative splicing
Dscam gene
- Most complicated gene
- Capable of producing 38,000 different proteins
- Through multiple exons
- Involved in nervous system
Give two examples of regulation of alternative splicing
Sex determination in drosophila
Generation of antibodies
Explain how sex determination in drosophila is an example of alternative splicing
Three genes key for sex determination: sxl, tra, dsx
In males
- sxl and tra are spliced to give rise to an inactive protein
- Dsx transcripts give rise to a male specific repressor protein that represses transcription from genes required for female development
In females
- A small amount of functional sx1 protein that binds to u2AF repressing splicing
- This feedback on the sxl transcripts to make more of itself and also binds to tra transcripts
- Results in a female specific isoform of dsx which represses transcription of male development genes
Explain how generations of antibodies is an example of alternative splicing
B lymphocytes produce two antibody isoforms
This antibody gene has two possible positions for cleavage and polyadenylation
When the cell produces the long transcript, the first stop codon is spliced out
- Results in the translation of a transmembrane domain
- When the cell switches t the short transcript, the splice acceptor is lost and the first stop codon us not lost
- This results in the antibody being secreted
- Alternative endings allow the gene to produce two different isoforms
How can alternative start sites give rise to different isoforms?
Leaky Scanning
- Sequences around the start help initiate translation. The optimal sequence is known as the Kozak sequence: ACCAUGG
- If the sequence is less than perfect, the small ribosome can scan past the first AUG and stop at a second or third AUG.
- These are all in the same reading frame, so the different isoforms of the protein differ only by the sequence of there N-terminus.
What is meant by the Kozak sequence?
The optimal sequence for initiation of translation:
ACCAUGG
How does eIF-4F affect alternative start sites?
High levels of eIF-4F in a cell favour the first AUG
Lower levels will favour the second or third AUG
How does HIV integrate its genome into the host?
Uses reverse transcriptase to convert their RNA into DNA and insert into host genome
How does HIV regulate its nuclear transport to ensure its unspliced RNA can leave the nucleus?
Full length RNA genome of HIV does to want t be spliced meaning it cannot leave the nucleus and er have a checking system that ensures unspoiled RNA cannot leave
- HIV therefore produces a protein REV which reenters the nucleus and binds to HIV introns and interacts with the nuclei pore to allow exit of surpliced RNA
- Can monitor levels of HIV infection by looking at levels of REV
What is a UTR?
Untranslated region of mRNA
- Found in all mRNA
- Can be 3’ or 5’
How can UTR’s be regulated?
Intermolecular base pairing within the UTR can form stem loops
Allows the UTRs to be recognised by cellular proteins and allowing transcription
Useful in development -forms gradients of RNA
Give an example of how UTRs can regulate translation
Iron homeostasis
- Regulated by ferritin and transferrin
- Ferritin is a protein that stores iron in the cell and thus increasing the iron available
- Aconitase acts as a switch - when iron is low aconitase binds to ferritin mRNA 5’ UTR and blocks translation stopping production of the iron storing protein. Aconitase also binds transferrin mRNA 3’ UTR which stabilises the RNA stopping degradation
How does iron and aconitase work in a negative feedback loop?
Iron binds to aconitase stopping it from binding to ferritin mRNA to reflow it’s production
Why is homeostasis of iron very rapid?
Because it works at the level of mRNA so doesn’t have to wait for transcription factors and other machinery
What is the role of the active form of eIF-2?
When eIF-2 is active it is bound to GTP
IT binds to Met tRNA and starts ribosome scanning
How can elf-2 and eIF-2B regulated translation?
When eIF-2 is active it is bound to GTP
Once hydrolysed it is bound to GDP
eIF-2b is required for the dissociation of GDP from eIF-2
The cell can interfere with this recycling by phosphorylating a position on eIF-2 so that it cannot be released from eIf-2B and translation is decreased
How can there be more than one reading frame on mRNA?
The presence of IRES changes the reading frame
What is an IRES?
An internal ribosome entry site
- Forms a stem loop that allows more than one reading frame
- Does this by initiating formation of the ribosome independent to the cap/polyA initiation complex
- eIf-4G is required for IRES based initiation and binds to the IRES stem loop
Why do viruses use IRES’s?
Because they an amplify the second reading frame by cleaving eIf-4G so it cannot interact with the cap and therefore favours the IRES
How are IRES’s uses in apoptosis?
eIF-4G is cleaved into a form that cannot bind eIF-4E, but still binds IRES
Certain genes required during cell death utilize IRES and they continue to be translated
How does RNA stability regulate transcription?
- The half-life of different mRNAs varies greatly from many hours to a few minutes
- Poly A tails start at about 200 in length, but an exonuclease chews them down to 30nt, at which point they are decapped and degraded.
- However, some mRNAs are re-adenylated in the cytoplasm to activate them or to extend half-life.
- Often factors that promote translation block degradation: DAN competes with eIF-4E for binding to the cap.
What is needed for many forms of non genetic analysis go gene function and what are the steps for its production?
Protein specific antibodies
- Makes lots of protein using vectors
- Antibody affinity purification
- Injection into an animal
Before you can generate a protein specific antibody, what is required?
Lots of purified protein of interest
How you can generate large quantities of your protein of interest?
- Use a plasmid vector that replicates in bacteria but also contains a promotor region
- Insert cDNA that codes for protein of interest into the plasmid vector
- Transform into competent bacteria
- The promotor region is a bacteriophage promotor meaning it can drive high levels of RNA synthesis
- The bacteriophages are so active that it would kill the cell so they must be inducible so can be turned on and off
- Bacteria are then lysed by adding detergent to break down cell walls and make a crude extract
Why must cDNA be inserted into the plasmid and not genomic DNA?
Because bacteria do not have introns
What is a bacteriophage?
A virus that specialises in infecting bacteria
How can an inducible promotor be turned on and off?
Chemicals or temperature shifts
What is a problem with using bacteria to generate a large amount of protein?
They don’t have the exact same folding chaperones so proteins may be folded slightly differently
This should be fine for most proteins though
How do we purify large amounts of protein from the crude extract?
Antibody-affinity purification
What is epitope tagging?
Allows us to rapidly purify our protein
Fuse our cDNA to epitope tag which is a DNA sequence that encodes for a short peptide that is specific to an already available antibody
Outline the steps in antibody-affinity purification
- Crude lysis of cDNA and tag
- Pour the crude in a tube full of beads which are coated in the antibody that recognises the epitope tag
- The cDNA/tag will bind to these antibodies and bacterial proteins will fall straight through
- Wash to remove non specific proteins
- Then add a buffer with pH 3 to disable the binding of the antibody to the tag so the protein of interest can be washed through -elusion
- Makes fractions of the protein which can then be analysed
After having large quantities of purified protein of interest, how do you make a protein specific antibody?
Inject the protein of interest into an animal (often a rabbit) over a three month period
Rabbit immune system will respond as its foreign and will produce antibodies which can then be purified from their blood
How can you tag antibodies?
Fluorescent dyes
Conjugated enzymes
How can fluorescent dyes be used to detect tagged antibodies?
Detection of the location using specific wave lengths of light
Can be done in a single cell or whole organism
Different colours provide high contrast so are easier to quantify
Give examples of enzyme conjugates?
Alakaline phosphatase - substrate turns blue
Horse radish peroxide - substrate turns brown
What is the advantage of using conjugated enzymes in antibody detection?
Enzyme detection can enhance sensitivity
How can you amplify the signal of antibody detection?
Antibody sandwich
- Primary antibody binds to protein
- Many secondary antibodies are added which can bind to the primary antibody amplifying the signal
Once you have a protein specific, how do you use antibodies?
- Chemically fix the animal tissue with formaldehyde to stabilise the structure
- Incubate with tagged antibody
- Antibody binds to target protein
- Wash off excess antibody and view using method of tagging
- Can be done in whole mount (whole organism) if small or sections of tissue if larger
What is the purpose of RNA in situ analysis?
It is a fast way of looking at expression but can only see RNA
What are the steps in RNA in situ analysis?
- cDNA cloned into plasmid
- In vitro RNA synthesis - plasmid DNA converted into antisense RNA - incorporating epitope tagged nucleotides
- Incubate embryo with antisense probe
- Antisense probe hybridises with the endogenous mRNA
- Wash of excess probe
Give the characteristics of green fluorescent protein (GFP)
Purified from jellyfish and was the first fluorescent protein discovered
A barrel shaped protein that emits green light with wavelength of 510mn if excited with light of wavelength 475nm
How do you generate a GFP transgenic line?
- Clone the entire gene with all of the regulatory elements into a plasmid
- Genetically engineer GFP onto the end of the last exon (gene fusion) or replace the gene (reporter construct)
- Integrate the GFP fusion gene back into the organisms genome - usually involves microinjecting a solution of the DNA into the one-cell zygote, DNA then randomly integrates into the genome.
What is a transgene?
An gene that has been transferred into the genome of one organism from an other
What are the uses of GFP transgenic lines?
Follow expression of gene
Localisation of protein
Behaviour of cells in vivo
High contrast between colours and black background - able to quantify position and levels of fluorescence
Who came up with the phylogenetic tree of animals?
Carl Linnaeus
What did genome sequencing show us about genes of different animals?
That most of the ones are the same in all animals investigated (house keeping genes)
How are proteins from different organisms compared?
Using programmes such as BLAST
- Align protein sequence and determine significant sequence homology
What is the rate of DNA sequence change between great apes and man?
the change in nucleotide sequence is about 1% every 10 million years
Give an example of how using the simplest model we can predict DNA sequence of common ancestors of animals
Highly conserved protein (FOXP2)
- has differences in the amino acid sequence at position 80, 303 and 325. Since mouse and chimp have 303 T, it is likely that their common ancestor also had a 303 T: since human and chimp have 80 D, it is likely that their common ancestor also had a 80 D
What is meant by parsimony?
For the analysis to work we assume the simplest model is true
What is meant by convergent evolution?
The similarity in base position between organisms occurred by chance making the ancestors more complex
- Could be due to selection pressures we are unaware of
How can we get a deeper understanding of evolution?
Molecular phylogeny is compared with morphological phylogeny and fossil records
FGFs can be split into 4 clusters based on protein sequence alignment, how many representatives does a Coina have?
Has single representatives in each of the 4 groups. This suggests that the common ancestor of the sea squirt and vertebrates had 4 FGFs
How do different types of FGF occur?
Gene duplication: changes in ploidy and local duplications
What are paralogues?
New copies of genes that arise in the genome
Once a chromosome or part of a chromosome has been duplicated, what occurs?
After a duplication, it is likely that the duplicate genes are at first redundant. This is thought to have played a major role during evolution, allowing for refinement of function or for a new function to evolve
How does a duplicated gene change to create a function?
1) pattern of expression
2) structural in the protein. Both small changes caused by point mutations and big changes caused by domain swapping.
What is thought to be the biggest driving force for morphological evolution in animals?
Changes in gene expression
How can change expression lead to morphological evolution?
Because enhancers can change easily
- eg. through non homologous recombination for example could bring a new enhancer close to gene
DNA sequence for TF binding sites is simple - - Relatively easy to add or delete sites by rearrangements, insertions, deletions or base pair substitution.
Give evidence that changes in the expression of single genes has played a major role in morphological evolution
Changes in expression of regulatory genes can correlate to morphological changes.
Hox C6 determines cervical vertebrae
- Hox C6 boundary is higher in mice then in chicks
- Expression of the gene C6 starts more posteriorly in chick – this correlates with a longer neck (more cervical vertebrae) and less chest (fewer thoracic vertebrae) than in mice
What is meant by master regulatory genes?
Regulate whole gene networks
How can you create ectopic organs just by changing the expression of a single gene?
Change master regulatory gene e.g.. ey
- Can create a working ectopic eye
What is meant by evolutionary robustness?
When an ectopic organ is still functional
e.g. ectopic eye can send signals to brain or ectopic leg will still move to try and walk
Why do crustaceans have legs on their abdomen but flies don’t?
Flies expressed Dlx in leg precursor cells and express Ubx in the abdomen
- Ubx represses Dlx stopping the development of legs on the abdomen
Crustaceans express both Dlx and Ubx in abdomen and thorax suggesting Ubx no longer acts as a repressor in crustaceans
- Crustacean Ubx has an antirepression motif that was lost in insects
What makes up the plasma membrane?
Lipids
– continuity and flexibility of the cell membrane
Proteins
– traverse the membrane, transport functions
Carbohydrates (sugars)
– cell protection and tagging
What are the three types of phospholipids in plasma membranes?
Phosphatidyl - ethanolamine
Phosphatidyl - serine
Phosphatidyl - choline
What are the essential properties of membranes?
Flexible
Seal interior from outside
Assymetric (sugars on the outer leaflet of the plasma membrane, negative charge on the inner leaflet)
What makes membranes flexible?
Fully saturated lipids are too rigid
Unsaturated fatty acids provides some disorder and therefore
flexibility to cell membranes
Several double bonds (= unsaturated) make fatty acids much more flexible
What is the role of cholesterol in the membrane?
Cholesterol helps to seal the plasma membrane preserving internal molecules
Why does the inside of a cell membrane have a negative charge?
Most intracellular molecules and vesicles are negatively charged and thus are intrinsically repulsed by each other and the plasma membrane
Why is Phosphatidylserine very important in cell membrane?
Phosphatidylserine is negatively charged and is strictly found on the inner surface of the cell membrane
Phosphatidylserine (PS) flips to the outer surface only upon apoptosis which takes place during cell death
Outline the receptor mediated endocytosis of cholesterol
- Cholesterol is transported in the blood as cholesteryl esters in the form of lipid protein particles know as low density lipoproteins (LDLs)
- When the cell needs cholesterol it synthesises LDL receptor membrane proteins
- When LDL binds with receptor, it causes calthrin coat to associate with membrane
- This causes invagination - forming coated vesicle containing LDL and receptor
- Vesicle becomes uncoated and fuses with an early endosome
- Proton pump acidifies the endosome thereby breaking LDL binding to its receptor
- The late endosome fuses with lysosomes freeing the cholesterol
How many binding sites does a LDL receptor have?
Two
- 1 extraceullar for LDL binding
- 1 clathrin coat binding site
What can mutation in LDL receptors lead to?
Atherosclerosis
- Mutations in LDL receptor account for familial cases of atherosclerosis, a cardiovascular disease
- Coated pit cannot form properly due to a mutation in the receptor and the inability of the receptor to interact with adaptor proteins
- This is due to accumulation of lipoproteins in blood and formation of plaques blocking blood arteries
How does clathrin help in the formation of vesicles?
It has a tri legged structure and forces membrane invagination
It shapes the vesicle and dynamin pinches it off the cell by hydrolysing GTP to GDP
What coats are used on vesicles in endocytosis in the ER and the Golgi?
Golgi - COP1
ER - COP11
Give an example of vesicle without clathrin
Phagocytosis
Outline the role of actin in phagocytosis
Bacterium binds to phagocyte inducing the cell to extend pseudopods that engulf the particle forming phagosome
Actin polymerisation shapes the pseudopods
To seal off the phagosome and complete engulfment, actin is depolymerised
The phagosome fuses with lysosomes destroying bacteria
What is autophagy?
Another degradation pathway in lysosomes to dispose of obsolete parts of the cells itself
The defective organelle is enclosed by a double membrane creating an autophagosome which then fuses with a lysosome
It then releases lipids and amino acids into the cytosol
What is the role of exocytosis?
Exocytosis is responsible for secretion of hormones, digestive enzymes,
recycling of plasma membrane receptors and neuronal communication
Give an example of regulated exocytosis
Release of highly-concentrated insulin from pancreatic beta cells
It happens only in response to high glucose
How were SNARE proteins discovered?
by researching exocytosis
How do SNARE proteins allow for the fusion of membranes?
The v-SNARE synaptobrevin is present on the vesicle
The t - SNAREs syntaxin and SNAP25 are present on the target cells
These SNAREs interact with each other causing their helical domain to wrap around each other forming a stable 4-helicle coiled coil
This interaction locks the membranes together
How many helicies do the different SNAREs contribute to the 4 helical coil?
SNAP25 contributes two
Syntaxin contributes one
VAMP contributes one
How does botulinum neurotoxin work?
Attacks SNARE proteins
What bacterial infections can cause neuromuscular paralysis?
Botulinum
- After consumption of contaminated food
Tetnus
- After skin cuts
What is the mechanism of action of BOTOX?
- Binds to gangliosides on neuronal membranes
- It enters the luminal space of recylic synaptic vesicles
- Following endocytosis, one subunit (SNARE protease) escapes the vesicle, enters the cytosol and cleaves SNARE proteins
- SNARE cannot support synaptic fusion anymore resulting in blockade of neurotransmission
- Re synthesis of SNARE can regain function
Which SNARE does botox cleave?
SNAP25
What can botox be used for?
Cosmetic - anti wrinkle
Muscular spasms
Dystonias
What are the types of protein trafficking?
Membrane protein synthesis
Protein tagging by sugars (glycosylation)
Protein activation by trimming (proteolytic processing)
Why do cells carry sugars on the outside of cells?
To protect themselves from harsh environment
and provide a tagging mechanism
Where are the different types of proteins made?
Cytosolic proteins are made in the cytosol
Membrane proteins and secretory proteins are made in the rough endoplasmic reticulum
Nuclear proteins are made on the outer nuclear membrane
What happens to proteins after synthesis?
Following synthesis, proteins move into smooth endoplasmic reticulum where lipids are made and vesicles form
Final addition of sugars and sorting takes place in Golgi
Proteases trim and activate hormones and enzymes during maturation of vesicles
What is a protein translocator?
A channel found in the ER membrane - move protein from ribosome to ER
How do newly synthesised peptides enter the ER lumen?
- The ER sequence is guided to the ER membrane by a signal recognition particle (in the cytosol) and its receptor (in the ER membrane)
- The SRP binds the ER signal sequence
- The SRP wraps around the large subunit of the ribosome and binds to the end of the newly formed ER signal sequence as it emerges from the ribosome. The other end of the SRP blocks the elongation factor in the ribosome, slowing down protein synthesis
- The SRP binds to the SRP receptor in the ER membrane brining the ribosome to the adjacent protein translocator in the same membrane
- The SRP and receptor are then released and the translocator transfers the growing peptide chain across the ER membrane
What happens if there is over 20 hydrophobic residues on a protein entering the ER?
It will become stuck in the membrane of the ER
What is responsible for removing the protein from the protein translocator into the ER?
Signal peptidase
What are the functions of the ER?
- The signal sequence is removed by a specific signal peptidase
- This is followed by conformational maturation
- Disulfide bridges are formed between cysteine residues to ‘solidify’ protein shape
- The protein is glycosylated by a standard carbohydrate chain (quality control)
How do ER enzymes return their after modifications?
ER-resident enzymes carry KDEL sequence
Why is the addition of carbohydrates to proteins important?
- Protein stability in the harsh extracellular environment
- Cell-cell recognition (subtle changes have major effects)
- Cross-species separation (humans use beta-galactose; other animals use alpha-galactose)
Give the steps in glycosylation of proteins
- Initial addition of carbohydrates starts in the ER and start as quality control tags
- The trimming and growth of carbohydrates then occurs in individual Golgi cisternae
- Each glycosylation step requires a separate Golgi compartment to keep the specific glycosylation enzymes away from each other
- The protein enters the Golgi to the cis Golgi network, where the proteins are sorted and phosphorylation of oligosaccharides on lysosomal proteins occurs
- It then moves into the cis cisterna, the medial cisterna and trans cisterna where are a series of glycosylations and modifications occur
- The protein then leaves the Golgi from the trans Golgi network after being fully modified
How are the limitations of organ transplantation cause by a sugar modification?
Human cells make beta galactose while other animals make alpha-galactose
Humans will produce antibodies against alpha-galactose and thus reject transplants
Animal-derived organs are rejected by the human organism
How are the limitations of organ transplantation being overcome?
Genetically-modified pigs lacking alpha-galactose
make organ transplantation possible
Give an example of protein glycosylation with a human population
Carbohydrates of Band 3 protein in erythrocytes determine human blood compatibility
What are the four blood groups?
O
A
B
AB
What determines blood groups?
Blood group is determined by a single terminal galactose residue
O - lacks a terminal Gal
A. - acetylated Gal
B - normal Gal
Give an example of protein trimming
Occurs prior to secretion
- Preproinsulin is cleaved to proinsulin in the ER
- Proinsulin is cleaved to insulin in the secretory vesicles
How is type 1 diabetes caused by by a failure of protein trimming?
- Misfolding of Proinsulin in the ER due to a mutation because the protease in secretory vesicle cannot cleave off the C-peptide
- Causes secretion of dysfunctional pro-insulin instead of insulin into blood
- Generation of antibodies against the pancreatic cells
- Destruction of pancreatic cells
- Causes an increased blood glucose concentration
Give an exmaple of how cleavage of polypeptide can lead to different hormones
Cleavage of opiomelanocortin can give rise to several hormones:
- ACTH and β-lipotropin are secreted by the pituitary gland
- β-endorphin is generated by neurons in response to exercise and stress.
What clinical syndromes can arise from mutations in an insulin receptor?
Leprechaunism
- Usually fatal within the first 2 years of life
- “elfin-like” facial appearance with protuberant ears and relatively large hands and feet. A decreased amount of subcutaneous fat and muscle mass is seen, and the skin is abnormal with increased hair growth.
Rabson-Mendenhall syndrome
- Survival into the second decade
- Skin and teeth abnormalities, hair overgrowth and pineal hyperplasia.
Type A insulin resistance
- Survival into middle age and beyond
Where is insulin produced?
Insulin is synthesised in beta cells of islets and is secreted into the blood stream
Give the structure of insulin
It is cleaved after translation to remove the inside so only the C and N-terminus are present. The two termini are connected by disulphide bridges which are form between cysteines in an oxidative (extracellular) environment
What are disulphide bridges used for?
Disulphide bridges are important in giving structure to extracellular proteins
What are the immediate effects of insulin?
Causes the uptake of glucose from the blood into the muscle cells and adipocytes to decrease glucose present in the blood
Takes less than a minute
What are the long term effects of insulin?
Causes an increased expression of liver enzymes that synthesize glycogen and increased expression of adipocyte enzymes that synthesize triacylglycerols
Because this works at the level of transcription it takes at least two hours for it to take effect
Can cause DNA modifications that can cause the effects to last for weeks
What pathways does insulin exert its action on?
RAS pathway
RAS indépendant signalling pathway
Outline the RAS pathway
- The insulin receptor is a dimer consisting of alpha and beta subunits. These subunits are synthesised as a single polypeptide that is cleaved into two fragments. These fragments are held together by disulphide bridges
- Insulin binds causing autophophorylation. The receptor is a kinase that can phosphorylate itself
- The phosphorylation is recognised by insulin receptor substrate (IRS) containing a phosphotryosine binding domain
- IRS binds to the insulin receptor and is phosphorylated by the receptor
- The phosphorylated IRS then initiates a Ras cascade causing a kinase pathway. Each kinase activates the next kinase
- This eventually activates a transcription factor through this phosphorylation causing gene expression
Outline the RAS independent signalling pathway
Insulin binds to the insilin receptor causing autophosphorylation
IRS recognises the phosphorylation and binds causing phosphorylation if its self
This creates a binding site for PI-3 kinase
PI-3 kinase is made up of two sub-units: P85 which has an SH2 domain and P110 which is a kinase
Upon binding to IRS, PI-3 kinase phosphorylates PI 4,5-bisphosphate and PI 4-phosphate to PI 3,4,5- trisphosphate and PI 3,4-biphosphate, respectively
This creates a docking site for PKB
PKB is held to the membrane allowing for its phosphorylation by membrane associates kinases such as PKD1
PKB undergoes a conformational change to become active and can then effect numerous proteins
What is the action of protein kinase B after the binding of insulin?
- Activates glycogen synthase by inhibiting GSK through phosphorylation. This allows glucose to be stored
- It phosphorylates Glut4, which is usually stored in vesicles in target cells, causing its release to the membrane. Allows glucose to be transported into the cells
- Phosphorylates FOXO, a transcription factor that activates PEPCK transcription, inhibiting it. PEPCK is an enzyme that is involved in glucose synthase. Inhibiting its transcription, lowers blood sugar
How can we identify genes that are activated by insulin?
Microarrays
PCR
Promotor bashing
What are the uses of PCR?
To amplify DNA
To clone DNA from minute samples
To quantify levels of RNA
Outline the steps in PCR (to amplify a specific sequence)
- Start with a double stranded DNA template
- Heat the template to cause the DNA to denature and form single stranded DNA
- Anneal two primers that are complementary to the right and left boundaries of the desired sequence. These primers must be on opposite strands and face each other
- DNA polymerase is then allowed to replicate each strand independently
- After the second cycle of PCR, there are four DNA strands produced, but not of the correct length
- After the third cycle, 8 strands are produced and two double stranded DNA of the correct length and sequence
- Repeat this process and you will eventually end up with millions of copies of pure target sequence
How is PCR been able to be developed?
This process was reliant on the discovery of heat stable DNA polymerase from thermos aquaticus bacteria (TAQ)
Allows us to put all the components into a machine that changes temperature, to allow for each stage, without having to stop after every cycle
Outline RT PCR
Reverse transcription polymerase chain reaction – RT PCR
- Isolate RNA from cells which can be transcribed into DNA using reverse transcriptase
- Amplify DNA using primers and DNA polymerase
What are the advantages of RT-PCR?
Can be used to investigate RNA which is specifically protein coding unlike DNA which has non-coding sequences (introns)
Outline qPCR
Quantitative PCR
- Make cDNA from tissue
- Perform PCR in presence of a fluorescent dye
- Measure the fluorescence at the end of each cycle
- After the 10th cycle start to detect fluorescence
- Establish standards of the amount of fluorescence should be seen for a certain number of copies of cDNA
- Compare the PCR fluorescence with the standards to find out the number of cDNA copies
Outline promotor bashing
- Make a transgene that uses a quantifiable reporter e.g. luciferase
- Luciferase produces a fluorescent substrate when activated
- Slowly delete parts of the promotor and test to see its responsiveness based on the illumination seen
- Can use this to find a critical region in the promotor that defines activity
- Can use this to determine which part of the promotor is required to bind insulin
How was it discovered that cancer derives from cells and is not a result of infection?
First had to understand the concept of cells a d tissues
Virchow (1858) said that all cells come from cells
This discovery allowed people to understand that this included tumours. All tumours are derived from cells
Define tumour
Tumours are created by cells that have lost the ability to assemble and create tissue of normal form and function. There are diseases of malfunctioning cells
Define hyperplasia
Tissue growth containing excessive numbers of cells
Define metaplasia
Tissue growth containing displacing but otherwise normal cells
Define dysplasia
Tissue growth where cells appear abnormal
Define neoplasia
Invasive, abnormal tissue growth
- Invasion of tissues
Why are neoplasia tumours usually associated with aggressive forms of cancer?
These types of cancer can’t easily be removed by surgery without removing healthy tissue
Can be fatal as unable to survive without the tissue that has been invaded
What is the hypothesis of tumour progression?
The more aggressive cancers tend to be further down the list of tumour types (neoplasm) suggesting a direction of travel as cancer cells emerge
How does histopathology allow us to investigate tumours?
It allows us to predict the common behaviours among diverse tumours despite radically different functions
What is the major risk factor of cancer?
Age
- 75% of 75 year olds, who do not die of cancer, have a tumour
- Different cancers have different risks and doesn’t always increase in age
- E.g. Hodgkin lymphoma – peaks in 60s and 70s
Are genetic or environmental factors a bigger cause of cancer?
Environmental
Give evidence for environmental factors causing cancer
Variation between specific groups
- In japan, people are likely to get stomach cancer but not prostate, colon and breast. Caucasian people have very high levels of prostate, breast and colon in comparison but lower stomach. Japanese people who migrated to Hawaii (culturally Caucasian) start to follow the patterns of them – environmental not genetic
Which cancers are known to have genetic factors?
- Retinoblastoma
- Wilms tumour
- APC
- Breast cancer
Is the incidence of cancer increasing?
- Cancer itself is not increasing but the population is ageing
- Cancer development increases with age so the incidence is increasing because people are living longer
What is the relationship between the log of the incidence of cancer and the log of age?
It is a straight line
y = mX+c
P(C1) ∝Age (they are proportional)
What is the probability of the changes required for tumour development and age?
P(T)=P(C1).(PC2).(PC3)…..(PCN) x A^N Where N is the number of changes, and A is age
Why does the relationship between cancer incidence age result in a straight line?
P(T)=P(C1).(PC2).(PC3)…..(PCN) x A^N equals
LogP(T )= nLogA + constant
This is the same is Y = mX + C
How many changes/mutations must occur for a tumour to develop according to the log equations?
6
The slope of the graph
Why do we not know which 6 mutations must occur to cause a tumour?
Genome instability causes cancer
When analysing of the tumour, the genome is very incoherent. Sequencing of a tumour genome will show lots of mutations (thousands) but these mutations are not necessarily all in the same cell.
Lots of different cells have lots of different genotypes and therefore phenotypes
How long does a cell cycle take?
20 hours
How has radioactivity been used to investigate what stage of the cycle cells are in?
- Add radiolabel to suspected interphase cells
- The radioactive phosphate emits beta particles which can be detected
- Incubate cells with radioactive phosphate and it will be taken up into nucleic acids as they have a sugar phosphate backbone
- However, they are only taken up into nucleic acid if DNA replication is occurring as that is when the incorporation of new phosphate occurs
- Wash out the free radioactive phosphate and expose these cells to a film then radioactivity will be detected.
- 35% of cells will incorporate as they are in interphase
How can you use the information from the radioactive phosphate to work out the time the cell spends in each part of the cycle?
Can use this percentage to calculate the time the cell spends in each stage of the cell cycle. It takes 20 hours for a cell to replicate so would only expect 5% of the cells to be in mitosis meaning it takes an hour for mitosis to occur
How can we investigate the cell cycle using variants of nucleotide precursors?
Chloro deoxyuridine is included into the DNA where thymine usually would be in a newly synthesised DNA strand
This can be detected by antibodies specific to chloro deoxyuridine and shows where DNA replication is occurring (green)
Can also detect DNA replication with iododeoxyuridine (red) and compare these two molecules using specific antibodies to see how the location of DNA replication varies throughout S phase
How can morphological markers me used to study the cell cycle?
Tubulin staining can be used to see what stage of mitosis cells are in as microtubules align the spindles at the midpoint of mitosis
How can functional in vivo assays be used to study the cell cycle?
Stain live embryos or add GFP to specific gene
Can see live how the cell cycle occurs
How can the results from functional in vivo assays be used to investigate the length of time in each stage of the cell cycle?
Can then use a flow cytometer to measure the content of the cells based on their fluorescence
A cell will either have a G1 content of DNA (normal) or twice that amount of DNA if the cell is in S phase
This results in the number of cells and how many of these cells are in which stages of the cell cycle
This can be used to work out how long the phases last based on how many of the cells are in each stage
What types of yeast have been used to study the cell cycle?
Fission yeast
Budding yeast
How have fission yeast been used to study the cell cycle?
Fission yeast only grow in one direction so can determine where the cell is in its cycle just be measuring its length. It is very precise
How have budding yeast been used to study the cell cycle?
Budding yeast divide by growing buds. The morphology of these buds can be used to see where the yeast is in the cell cycle
How to geneticists cause mutations of genes involved in the cell cycle without completely arresting the cycle?
Use temperature sensitive mutants which get stuck at a critical time in the cell cycle but only at specific temperatures (usually higher)
At some temperatures, the mutant will be able to complete the cell cycle normally.
How do temperature dependant mutations occur?
The mutation we have introduced creates a gene product that is functional at one temperature and not at others (protein loses its shape)
Why are temperature dependant mutants required when studying the cell cycle?
If the cell cycle is disrupted then cannot get large colonies of the mutants to investigate
Why are certain species of toads a good model organism?
If injected with progesterone, the frog will lay eggs at any time of the year
Large eggs
What Is the role of cyclins in the cell cycle?
Cyclins are proteins that are expressed at different levels during the cell cycle
When present, cyclins bind to specific kinases (called cyclin dependant kinases, Cdks)
CDKs phosphorylate many proteins that are specific to certain stages of the cell cycle. This works as a molecular switch to change the proteins function
How is the specificity of the cell cycle maintained in mammals?
There are specific cyclins and Cdks bound which target specific substrates relevant to a specific phase of the cell cycle
Describe the overall expression of cyclins in the cell cycle?
They increase and then are destroyed by proteolytic destruction
What did Rao and Johnson do to investigate the control of the cell cycle?
They infected cells at different stages in the cell cycle with Sendai virus, which then fused cells forming hetertropic fusions
What did Rao and Johnson observe?
- When you fuse a mitotic cell to a cell is G1, you get an induction of condensation
This occurs no matter where in the cell cycle that cell is.
When mitotic cell and G2 cell fused, there is better condensation. If joined with an S phase, the chromosomes are condensed but look broken because during replication chromosomes must be more open.
What did Rao and Johnson conclude from their experiment?
That there is something in mitotic cells which is dominant and capable of causing condensation of chromosomes anywhere in the cell cycle
What did Masui do to investigate the control of the cell cycle?
- He looked at the physiology of oocytes.
- When the egg is laid, it is in meiotic metaphase but before it is laid the cell is arrested in G2 phase
- The top of an oocyte is darker then the bottom. However, when the oocyte enters meiosis a small white spot appears on the dark side of the cell
- Masui took some cytoplasm from eggs in meiotic metaphase and injected it into oocytes that were arrested in G2 phase
The oocytes immediately went into M phase and little white spot appeared
What did Masui conclude from his experiment?
There must be a factor that causes oocytes to enter metaphase
- Maturating promoting factor (MPF).
What did a post doc of Masui do and conclude about the cell cycle?
Completed a biochemical assay to identify MPF
- Produced pure fractions which seemed to have MPF activity
- Two fractions which seemed to be consistent
- Protein kinase activity associated with one of these fractions
- Completed an autoradiogram showing that these fractions, when given radioactive ATP, will transfer the radioactive phosphate to a target histone. They concluded that MPF was two proteins working together and has protein kinase activity against Histone H1
What did Hunt do to investigate the cell cycle?
He studied protein synthesis in sea urchin eggs
He noticed that there was a protein in sea urchin eggs that were synthesised and destroyed
if you plot the intensity of these proteins, it was in synchronous with the cell division in sea urchin eggs, implying a link between them.
He named these proteins cyclins
He then purified the mRNA driving the synthesis of these proteins allowing their identification
What did Nurse do to investigate the cell cycle?
Investigating temperature sensitive mutants, in this case Pombe yeast. These dividing yeasts were stained to see the nucleus and cleavage during mitosis
What did Nurse observe and conclude from his experiment?
In the temperature sensitive mutants, which were arrested in G2, Cdc2 and Cdc25, the cells were much longer then in the wild type.
n the mutant Wee1 the cells were much smaller.
Genetic analysis of these revealed that Cdc2 was the central regulator of mitosis so that was when it wasn’t functional cells kept growing in G2.
Cdc25 was a positive regulator of mitosis and Wee1 is a negative regulator of mitosis
How was Cdc2 indentified?
A library of wild type genes was added to the mutant phenotype, allowing the phenotypes rescue. Extract the plasmids out of the yeast and sequence the DNA, you can find the wild type gene that rescued this phenotype (it will not be temperature dependant)
What was interesting about the identity of Cdc2?
Shared homology to a protein purified from frog eggs, showed it was evolutionary conservation
What would happen if you were to implant the human homolog of Cdc2, Cdk1, into a yeast Cdc2 mutant?
It would rescue the phenotype
- Shows evolutionary conservation
How is the specificity of the cell cycle ensured in yeast?
There is only one kinase in yeast. The specificity of the phases of the cell cycle the cyclins drive, therefore, is entirely determined by the cyclin that it is bound to
How many replications of origins are there in mammals and bacteria?
Tens of thousands in mammals
One in bacteria
What are pre-replicative protein complexes?
The origins of replication are recognised by pre-replicative protein complexes
They bind to the relevant sequence in G1 and act as markers for replication
In S phase, the pre-replicative complexes are displaced and destroyed.
How do we know that S phase only happens once?
Put DNA into frog extracts, the DNA will undergo semi conservative replication
In an extract, where you don’t allow the cell to enter mitosis, then DNA is only replicated once
How did we investigate why S phase only happens once?
- Centrifuged out the nuclei from the extract and put them in fresh extract
Still do not replicate - Put the nuclei in detergent, the DNA replicates once
- This is because the pre-replicative complexes are present in the cytoplasm but excluded from the nucleus
- In mitosis, the nuclear envelope breaks down and these proteins can enter the nucleus
- If added to detergent, the nuclear envelope becomes more permeable and these complexes can enter the nucleus
What is Cdt1?
A licensing factor that is a regulatory component of DNA replication that binds to these pre-replicative complexes. It allows once round of DNA replication and is then destoyed
This stops multiple replications
How has the discovery of Cdt1 helped in the diagnosis of cancer?
Antibodies that recognise the pre-replication complex and Cdt1 are profoundly useful for detecting malignant cells in cervical samples. E.g. smear tests
How are Cdks controlled?
CDK regulators include activators, mainly the cyclins, and inhibitors, generically known as CKIs (CDK kinase inhibitors). Inhibitors mainly operate in G1 and S
