BMS238 Cell and Molecular Biology Flashcards

Question

How can alpha helices interact with each other?

Answer 1

Double or triple coiled coil | - Coil together alpha helices with their hydrophobic side chains facing inwards creating a hydrophobic core

Answer 2

In elongated fibrous proteins that need the ability to form cloths

Answer 3

Circular Dichroism (CD) X-ray Crystallography Nuclear Magnetic Resonance (NMR) Electron Microscopy

Answer 4

You need to purify the protein of interest | into its purest form.

Answer 5

To estimate the secondary structure of a protein?

Answer 6

- 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

Answer 7

Can be used at different temperatures providing us with information regarding the proteins stability – the less stable, the quicker the structure will be lost

Answer 8

Calmodulin is a small calcium-binding protein which activates protein kinases and other proteins

Answer 9

There are two globular domains each with two calcium binding sites - total of four Sites occur in the loop region connecting two alpha helices

Answer 10

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

Answer 11

NMR spectroscopy is useful to understand dynamics of protein structures

Answer 12

The protein of interest needs to be highly pure and labelled with isotopes such as 13C or 15N

Answer 13

- 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

Answer 14

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

Answer 15

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

Answer 16

Transmission electron microscopy | Cryo-Electron microscopy

Answer 17

To study large protein structures

Answer 18

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

Answer 19

- 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

Answer 20

- Cheap, Quick, no size limit | - Limited dynamics, low resolution, secondary structure only

Answer 21

- High resolution, no size limit | - Expensive, slows down due to crystal formation

Answer 22

- Reasonable resolution, good dynamics | - Size limit of 50kDa

Answer 23

- No size limit - large structures - Quite expensive, medium speed - No dynamics

Answer 24

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

Answer 25

- 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

Answer 26

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

Answer 27

Velocity sedimentation | Equilibrium sedimentation

Answer 28

- 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

Answer 29

- 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

Answer 30

- 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

Answer 31

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

Answer 32

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

Answer 33

- 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

Answer 34

- 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

Answer 35

Cation exchange - CM carboyx-methyl Anion exchange - DEAE diethylaminoethyl

Answer 36

- 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

Answer 37

Using a mixture of ion exchange, gel filtration and affinity chromatography

Answer 38

Sodium dodecyl sulphate polyacrulamide gel electrophersis

Answer 39

- 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

Answer 40

- 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

Answer 41

Denatures the protein structure due to its CH group | Breaks down polypeptide chains into smaller fractions which can then be investigated separately

Answer 42

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

Answer 43

- 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

Answer 44

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

Answer 45

H202 addition produces lots of light can look via X Ray

Answer 46

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

Answer 47

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

Answer 48

Because it allows identification and sequencing of tiny changes in proteins

Answer 49

The presence of a P04 group increases the mass of serine by 95 daltons so a fraction will change if labelled with a phosphatase

Answer 50

Serine Threonine Trysoine Because they have hydroxyl groups

Answer 51

The term Proteomics refers to the analysis of complete protein content in a living system, including post-translationally modiﬁed proteins and alternatively spliced variants

Answer 52

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.

Answer 53

Hydroxylation Methylation Acetylation Lipids

Answer 54

Adds an OH group | +17 Daltons

Answer 55

Addition of CH3 group +15 Daltons Has an important role in epigenetics

Answer 56

Addition of CH2CH3 group +27 Daltons Role in epigenetics

Answer 57

Varies depending on the modification | - Usually +2000 Daltons and higher

Answer 58

1902 by Boveri and Sutton | Morgan discovered that chromosomes are the locations of genes in 1915

Answer 59

DNA and proteins

Answer 60

- 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

Answer 61

No small circular chromosomes are found in mitochondria and chloroplasts

Answer 62

The organised representation of all chromosomes in a eukaryotic cell and metaphase

Answer 63

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

Answer 64

Normal running of cell - housekeeping functions | Can see that each chromosome occupies a distinct subnuclear territory in the interphase nucleus

Answer 65

Active genes are in the centre and inactive are in the peripheral There are some exceptions but this is generally the pattern

Answer 66

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

Answer 67

A highly coiled fibre of chromatin

Answer 68

Resembles beads on a string where the beads are nucleosomes

Answer 69

It is a supercoiled array of nucleosomes

Answer 70

DNA wrapped around a protein core

Answer 71

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

Answer 72

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

Answer 73

Rich in basic amino acids such as lysine

Answer 74

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

Answer 75

Because the sequences facilitate reliable and complete DNA replication, segregation of duplicated chromosomes during cell division

Answer 76

DNA sequences at ends of linear chromosomes: maintain chromosomal integrity

Answer 77

DNA sequence where DNA replication is initiated

Answer 78

DNA sequences on which kinetochore assembles and mediates chromosome segregation at mitosis and meiosis

Answer 79

Protein complex that binds microtubules in the mitotic spindle

Answer 80

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

Answer 81

In the centre of the nucleus at the end of microtubule attachments

Answer 82

Alpha satellite DNA repeats which form condensed heterochromatin with histone octamers containing unusual subunits

Answer 83

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

Answer 84

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

Answer 85

Ensure the right pairings of sister chromatins - allow them to be next to each other

Answer 86

In yeast the kinetochore is a basket that links a single nucleosome of centromeric chromatin to a single microtubule

Answer 87

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

Answer 88

A few % DNA transposon 10% retroviral like elements 30% non retroviral polyA rectotransposons All are genetically mobile and can relocate on the genome

Answer 89

occupy a few% of the eukaryotic genome | - DNA sequences that are bound by short repeated sequences

Answer 90

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

Answer 91

Babara Mcclintock - 1952

Answer 92

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

Answer 93

30% of eukaryotic genome - Most abdudent type of transposons in the vertebrate genome - Replicate via RNA intermediate

Answer 94

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

Answer 95

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

Answer 96

Semi conservative replication | - Each strand is used to make a daughter strand

Answer 97

5' - Phosphate present on 5' carbon of the deoxyribose sugar 3' - OH present on the 3rd carbon

Answer 98

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

Answer 99

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

Answer 100

Where the DNA strands are separated by DNA Helicase so that DNA synthesis can be initiated

Answer 101

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

Answer 102

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

Answer 103

DNA primase synthesises a RNA primer from which DNA polymerase can extend from

Answer 104

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

Answer 105

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

Answer 106

``` 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 ```

Answer 107

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

Answer 108

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

Answer 109

The single stranded DNA may start to fold back on themselves causing them to become double stranded

Answer 110

Using single stranded binding proteins | - They keep the DNA strands straight to stop them from folding back on its self and becoming doubled stranded

Answer 111

``` 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 ```

Answer 112

``` - 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 ```

Answer 113

They are specific DNA sequences that direct the initiation of DNA replication by recruiting Replication Initiator proteins

Answer 114

Autonomously Replicating Sequence (ARS) Elements

Answer 115

DNA sequences near to LMNB2, MYC, HBB. But also defined by chromatin structure (e.g. nucleosome free region), rather than a specific DNA sequence.

Answer 116

1. Replicator Selection - formation of a pre-Replicative Complex (pre- RC) - occurs in G1 phase 2. Origin Activation - unwinding of DNA and recruitment of DNA Polymerase - occurs in S phase

Answer 117

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

Answer 118

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

Answer 119

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

Answer 120

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

Answer 121

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

Answer 122

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

Answer 123

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

Answer 124

No chemical bonds are formed, just multiple weak interactions

Answer 125

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

Answer 126

- Leucine zipper motifs - Zinc fingers - Basic helix-loop-helix (bHLH), - β-sheet

Answer 127

Increase the number of DNA binding motifs they have

Answer 128

- 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

Answer 129

Structurally - Zn2+ - Zinc finger Regulatory - Ca2+ - Calmodulin Catalytic e.g.. Zinc, iron and copper

Answer 130

- 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

Answer 131

Because one zinc finger only interacts with two nucleotide interactions - not strong so use multiple zinc fingers

Answer 132

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

Answer 133

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

Answer 134

To confirm DNA binding

Answer 135

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

Answer 136

Binds zinc to DNA

Answer 137

Protein - DNA binding

Answer 138

Binds calcium or magnesium in structural or signalling mode

Answer 139

Binds phosphorylated proteins

Answer 140

Binds proline rich motifs

Answer 141

Binds phosphorylated lipids

Answer 142

- SH2 domain - binds phosphorylated tyrosine (Grb2 protein) - SH3 domain - binds proline-rich motifs (Grb2 protein) - PH domain - binds phosphoinositide lipids (IRS1 and Sos proteins)

Answer 143

- 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

Answer 144

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

Answer 145

Protein tyrosine kinase Src

Answer 146

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

Answer 147

- 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.

Answer 148

Biochemically - Centrifugation, chromatography, pulldown reactions Structurally - X ray crystallography, NMR, electron microscopy

Answer 149

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

Answer 150

- 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

Answer 151

Glutathione-S-transferase (GST) | Hexa-histidine (6xHis)

Answer 152

HA peptide YPYDVPDYA Myc peptide EQKLISEEDL Flag peptide DYKDDDDK

Answer 153

- 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

Answer 154

To identify interacting sequences

Answer 155

- 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.

Answer 156

To measure the strength of protein interactions

Answer 157

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

Answer 158

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)

Answer 159

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

Answer 160

Kon[A][B] = Koff[AB] [AB]/[A][B] = Kon/Koff = K = Equilibrium constant

Answer 161

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

Answer 162

Oxidation, hydrolysis or uncontrolled random | methylation of any of the four nucleotides

Answer 163

Change the bases coding properties, remove a base from the DNA and actually fragment the DNA by breaking the phophodiester backbone

Answer 164

Hydrolytic deprurination and deamination of bases

Answer 165

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

Answer 166

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

Answer 167

Base Excision repair

Answer 168

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

Answer 169

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

Answer 170

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

Answer 171

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

Answer 172

Uvr proteins: | UvrA, UvrB, UvrC, UvrD

Answer 173

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

Answer 174

Because it could lead to large fragments of chromosomes being lost

Answer 175

Ionising radiation

Answer 176

1. Non-homologous End Joining | 2. Homologous Recombination (HR)

Answer 177

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

Answer 178

May join the wrong fragments together which can lead to loss of DNA fragments

Answer 179

The last line of defence in DNA repair | - It makes use of information that is in the undamaged homologous chromosome - high accuracy

Answer 180

In other forms of repair where there is a partial single stranded section of DNA - vulnerable to stress so may break completely

Answer 181

- 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

Answer 182

- BRCA2: breast, ovarian and prostate cancer - ATM: Ataxia telangiectasia – leukaemia, lymphoma - Fanconi Anaemia – complicated with leukaemia: 13 different FANC genes

Answer 183

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

Answer 184

The crossing over of one strand from one helix forming base pairs with the complementary strand of the other helix

Answer 185

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

Answer 186

DNA polymerase uses strands from both chromatids as templates to fill in self created breaks in meiosis

Answer 187

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

Answer 188

Typically one division every 24hrs in proliferating mammalian cells. Other cells do not divide once they are born and last for many years.

Answer 189

G1 - Gap 1 S - DNA replication G2 - Gap 2 M.- Nucleair division

Answer 190

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

Answer 191

Fission and budding

Answer 192

Rapid division rate <1hr Cell cycle control genes are highly conserved Yeast can be grown as haploids or diploids

Answer 193

- 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

Answer 194

Cell division cycle genes

Answer 195

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

Answer 196

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.

Answer 197

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?

Answer 198

- 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.

Answer 199

Wee1 kinase phosphorylates Cdks to inactive them | Cdc25 is a phosphatase that removes the phosphate group and activates Cdks

Answer 200

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

Answer 201

A protein that holds together sister chromatids

Answer 202

- 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)

Answer 203

Crossing over and segregation - DNA replication - Homologue pairs line up on spindle and are pulled apart

Answer 204

Resembles mitosis | Main difference to mitosis is that cells in meiosis II are haploid not diploid

Answer 205

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

Answer 206

For evolution

Answer 207

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.

Answer 208

Usually increased transcription means increased expression

Answer 209

RNA may be transcribed but not translated into a protein | Protein may be readily degraded

Answer 210

DNA protein interactions are essential for regulation of transcription

Answer 211

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

Answer 212

Lysine or Arginine

Answer 213

Reaches into the major groove of the DNA and forms two hydrogen bonds between asparagine and adenosine

Answer 214

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

Answer 215

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

Answer 216

By the frequency of each bases at each position A consensus sequence (average) Sequence logos

Answer 217

A protein structure that serves a purpose?

Answer 218

Helix turn helix Zinc finger Leucine zipper Helix loop helix

Answer 219

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

Answer 220

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

Answer 221

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)

Answer 222

Related to leucine zipper but has loops which enables more flexibility

Answer 223

The cooperatively of DNA binding motifs increases binding strength

Answer 224

DNAse I footprinting | EMSA - electrophoretic mobility shift assay

Answer 225

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

Answer 226

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

Answer 227

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)

Answer 228

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

Answer 229

A binding site for transcriptional activator | Are promiscuous - activate any genes in that area

Answer 230

Binding site for transcriptional repressor

Answer 231

Prevent the enhancer for activating all of the genes

Answer 232

An input that alters gene expression | - Each switch is responding to extrinsic or intrinsic regulation

Answer 233

Protein synthesis Ligand binding Protein phosphorylation Addition of second subunit

Answer 234

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

Answer 235

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

Answer 236

In the germ line | - May persist for the entire life of a somatic cell but can be erased in germ line

Answer 237

Different cell fates during development are the end of distinct journeys through an epigenetic landscape - Encounter different signals

Answer 238

The N-terminal lysine-rich tails of core histones project radially from the nucleosomal core and are covalently modified

Answer 239

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

Answer 240

Responsible for acetylation of lysine resides in core histones

Answer 241

Responsible for methylation of lysine and arginine

Answer 242

``` Histone acetyltransferases - can modify many different lysine residues in core histones Histone methyltransferases (HMTs) - highly specific so requires specific HMTs for specific lysine residues ```

Answer 243

Remove the acetylation and methylation of lysine residues

Answer 244

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

Answer 245

Exclusively activates transcription on whatever lysine it occurs

Answer 246

Acetylation | - Can exclude the action of methylation on the same lysine that would have lead to transcriptional repression

Answer 247

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

Answer 248

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

Answer 249

On lysine residues 27 and 9 on histone 3 - Create binding sites that contain chromodomains - These chromodomains bind and cause transcriptional repression

Answer 250

They only bind to methylations on specific lysine residues - 9 and 27

Answer 251

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

Answer 252

An epigenetic code that lies on top of the genetic code and governs when and where genetic information is expressed

Answer 253

- 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

Answer 254

- 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

Answer 255

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

Answer 256

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

Answer 257

Polycomb repressive complex 1 - Contains a chromodomain containing protein (called polycomb) at its core - Recognises lysine 27 on histone H3

Answer 258

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

Answer 259

Addition of methyl groups to cytosine residues

Answer 260

They physically interact with each tiger and mutually reinforce each other effects

Answer 261

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

Answer 262

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

Answer 263

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

Answer 264

1944 - Avery provides evidence that DNA carries genetic information 1953 - Watson, Crick, Franklin - DNA structure 1966 - Nirenberg, Ochoa, and Khorana elucidate the genetic code.

Answer 265

Three bases that encode an amino acid

Answer 266

Amino acids are specified by more than one amino acid (61 codons, 20 amino acids)

Answer 267

Where you start reading the codon from | - Three possible ones

Answer 268

By a start codon - where translation always starts | AUG (methionine)

Answer 269

UAA, UAG, UGA

Answer 270

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

Answer 271

Not 1:1 | - eg. there are 6 codons for serine but only 3 tRNAs

Answer 272

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

Answer 273

31 tRNAs for 61 codons

Answer 274

- 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

Answer 275

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

Answer 276

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

Answer 277

The ribosome

Answer 278

- 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.

Answer 279

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

Answer 280

Help translation and improve accuracy (EF-1)

Answer 281

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.

Answer 282

It is an RNA not a protein

Answer 283

A non protein based enzyme | - The ribosome is a ribozyme

Answer 284

Large subunit rRNAs form a massive structure that contains most of the catalytic activity including that of the petidyl transferase.

Answer 285

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

Answer 286

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

Answer 287

Yes multiple ribosomes can bind | - ribosomes are spaced about 80 nucleotides apart on a polysome

Answer 288

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.

Answer 289

Folding of the protein begins immediately after leaving the ribosome

Answer 290

Many proteins initially fold into roughly the correct confirmation. This is called a molten globule

Answer 291

As the protein exits it folds rapidly putting hydrophobic side chains in the middle to achieve a lower energy state.

Answer 292

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.

Answer 293

By molecular chaperones | - Reverse incorrect steps

Answer 294

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

Answer 295

The hsp70 class work directly on proteins as they exit the ribosome, binding to exposed hydrophobic amino acids

Answer 296

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

Answer 297

1/3 - due to misfolded | - Are therefore polyubiquitinated and targeted for destruction in the proteasome

Answer 298

CJD, Huntington’s disease and Alzheimer’s disease are all associated with large, extracellular protein aggregates.

Answer 299

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

Answer 300

Transcription is the process of RNA synthesis from a DNA template.

Answer 301

``` 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 ```

Answer 302

- Contains ribose (instead of deoxy-ribose) - Contains Uracil instead of Thymine - Synthesised as a single strand - RNA is very unstable

Answer 303

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

Answer 304

Stem-loop structure | - Formed due to base pairing

Answer 305

When the secondary structure folds into a there dimensional structure

Answer 306

Perform RNA synthesis

Answer 307

``` RNA polymerase I - ribosomal RNA RNA polymerase II - protein-encoding genes RNA polymerase III - tRNA, small nuclear RNA, and 5S rRNA ```

Answer 308

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

Answer 309

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

Answer 310

Chromosomes are too large to spin during replication so become knotted

Answer 311

Topoisomerases release supercoils to allow progression

Answer 312

Has specific promotor sequences that act as binding sites | - eg. TATA

Answer 313

- 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.

Answer 314

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

Answer 315

- 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.

Answer 316

The spliceosome

Answer 317

A nuclear complex made of about 150 proteins + 5 RNAs | Responsible for mRNA splicing

Answer 318

- Recognize 5’donor and branch sites - Bring sites together - Catalyzes RNA cleavage

Answer 319

- 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

Answer 320

- 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

Answer 321

They cut DNA into manageable sizes - restriction fractions

Answer 322

- 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

Answer 323

Using gel electrophoresis

Answer 324

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

Answer 325

Agarose gel | - Has a well (dip) in it where the DNA is added

Answer 326

Because DNA in solution is transparent so requires a dye that will fluoresce when it is bound to DNA

Answer 327

Joins DNA to create recombinant DNA

Answer 328

They can ligate because they can hybridise - the sticky ends are complementary to each other

Answer 329

SpeI and XbaI | - Their overhangs are cohesive

Answer 330

Plasmids are small, circular, extra-chromosomal DNA that occur naturally in bacteria.

Answer 331

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

Answer 332

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)

Answer 333

<30 kilo bases of DNA

Answer 334

<300 kilobases

Answer 335

< 3 megabases

Answer 336

Putting the DNA into the bacteria

Answer 337

Competent bacteria are bacteria that are ready to take up new DNA

Answer 338

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

Answer 339

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

Answer 340

Making a library of genomic clones or CDNA

Answer 341

Contains all the regulatory sequences as well as just coding DNA - Allows the study of transcriptional information

Answer 342

Only contains genes that are expressed as cDNA is made from RNA

Answer 343

- 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

Answer 344

Only the genes that are expressed in the original tissue (as opposed to genome which is all genes)

Answer 345

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

Answer 346

Purify out genomic DNA and make restriction digest | Represents the entire genome

Answer 347

- 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

Answer 348

Because it is missing an OH at the 3' end meaning another base cannot be added

Answer 349

While the sequencing gel is running, an electronic camera takes pictures of bands and measures intensity - can read 1000s of nucleotides

Answer 350

Progressive sequencing or shotgun sequencing

Answer 351

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

Answer 352

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

Answer 353

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

Answer 354

3. 2kb | - therefore cannot use dideoxycytidine terminator sequencing

Answer 355

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)

Answer 356

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

Answer 357

Microarrays allow us to compare the transcriptomes of different tissues to each other, eg normal liver tissue to cancerous liver tissue

Answer 358

It is small scale, fast and automated

Answer 359

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

Answer 360

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

Answer 361

- Making a library of cDNA clones - Making a library of genomic clones - Microarrays

Answer 362

Mouse genetics - Gene replacement - Gene knock out

Answer 363

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.

Answer 364

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.

Answer 365

- 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.

Answer 366

To identify the knock out

Answer 367

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

Answer 368

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)

Answer 369

1) randomly mutate the genome 2) look for interesting phenotypes in the offspring 3) identify the gene that causes the defect

Answer 370

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

Answer 371

In forward genetics we start with a phenotype to find the gene In reverse genetics we know the gene and we want the function

Answer 372

``` Positional cloning (linkage analysis) - A way of using a genetic map to find the mutation and identify the gene ```

Answer 373

- 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

Answer 374

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

Answer 375

A technique that allows mutations to be sorted into distinct groups that correspond to each individual genes

Answer 376

Used to identify genes

Answer 377

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

Answer 378

(Number of recombinant gametes/total number of gametes) x100

Answer 379

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.

Answer 380

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.

Answer 381

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.

Answer 382

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.

Answer 383

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

Answer 384

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

Answer 385

Different forms of proteins that can be formed by exactly the same genes

Answer 386

Alternative splicing - Optional exon - Optional intron - Mutually exclusive exons - Internal splice site

Answer 387

40% of drosophila genes and 75% of human genes

Answer 388

The sequence added/taken away is divisible by three

Answer 389

Dscam gene - Most complicated gene - Capable of producing 38,000 different proteins - Through multiple exons - Involved in nervous system

Answer 390

Sex determination in drosophila | Generation of antibodies

Answer 391

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

Answer 392

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

Answer 393

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.

Answer 394

The optimal sequence for initiation of translation: | ACCAUGG

Answer 395

High levels of eIF-4F in a cell favour the first AUG | Lower levels will favour the second or third AUG

Answer 396

Uses reverse transcriptase to convert their RNA into DNA and insert into host genome

Answer 397

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

Answer 398

Untranslated region of mRNA - Found in all mRNA - Can be 3' or 5'

Answer 399

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

Answer 400

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

Answer 401

Iron binds to aconitase stopping it from binding to ferritin mRNA to reflow it's production

Answer 402

Because it works at the level of mRNA so doesn't have to wait for transcription factors and other machinery

Answer 403

When eIF-2 is active it is bound to GTP | IT binds to Met tRNA and starts ribosome scanning

Answer 404

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

Answer 405

The presence of IRES changes the reading frame

Answer 406

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

Answer 407

Because they an amplify the second reading frame by cleaving eIf-4G so it cannot interact with the cap and therefore favours the IRES

Answer 408

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

Answer 409

- 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.

Answer 410

Protein specific antibodies - Makes lots of protein using vectors - Antibody affinity purification - Injection into an animal

Answer 411

Lots of purified protein of interest

Answer 412

- 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

Answer 413

Because bacteria do not have introns

Answer 414

A virus that specialises in infecting bacteria

Answer 415

Chemicals or temperature shifts

Answer 416

They don't have the exact same folding chaperones so proteins may be folded slightly differently This should be fine for most proteins though

Answer 417

Antibody-affinity purification

Answer 418

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

Answer 419

- 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

Answer 420

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

Answer 421

Fluorescent dyes | Conjugated enzymes

Answer 422

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

Answer 423

Alakaline phosphatase - substrate turns blue | Horse radish peroxide - substrate turns brown

Answer 424

Enzyme detection can enhance sensitivity

Answer 425

Antibody sandwich - Primary antibody binds to protein - Many secondary antibodies are added which can bind to the primary antibody amplifying the signal

Answer 426

- 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

Answer 427

It is a fast way of looking at expression but can only see RNA

Answer 428

- 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

Answer 429

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

Answer 430

1. Clone the entire gene with all of the regulatory elements into a plasmid 2. Genetically engineer GFP onto the end of the last exon (gene fusion) or replace the gene (reporter construct) 3. 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.

Answer 431

An gene that has been transferred into the genome of one organism from an other

Answer 432

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

Answer 433

Carl Linnaeus

Answer 434

That most of the ones are the same in all animals investigated (house keeping genes)

Answer 435

Using programmes such as BLAST | - Align protein sequence and determine significant sequence homology

Answer 436

the change in nucleotide sequence is about 1% every 10 million years

Answer 437

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

Answer 438

For the analysis to work we assume the simplest model is true

Answer 439

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

Answer 440

Molecular phylogeny is compared with morphological phylogeny and fossil records

Answer 441

Has single representatives in each of the 4 groups. This suggests that the common ancestor of the sea squirt and vertebrates had 4 FGFs

Answer 442

Gene duplication: changes in ploidy and local duplications

Answer 443

New copies of genes that arise in the genome

Answer 444

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

Answer 445

1) pattern of expression | 2) structural in the protein. Both small changes caused by point mutations and big changes caused by domain swapping.

Answer 446

Changes in gene expression

Answer 447

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.

Answer 448

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

Answer 449

Regulate whole gene networks

Answer 450

Change master regulatory gene e.g.. ey | - Can create a working ectopic eye

Answer 451

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

Answer 452

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

Answer 453

Lipids – continuity and flexibility of the cell membrane Proteins – traverse the membrane, transport functions Carbohydrates (sugars) – cell protection and tagging

Answer 454

Phosphatidyl - ethanolamine Phosphatidyl - serine Phosphatidyl - choline

Answer 455

Flexible Seal interior from outside Assymetric (sugars on the outer leaflet of the plasma membrane, negative charge on the inner leaflet)

Answer 456

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

Answer 457

Cholesterol helps to seal the plasma membrane preserving internal molecules

Answer 458

Most intracellular molecules and vesicles are negatively charged and thus are intrinsically repulsed by each other and the plasma membrane

Answer 459

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

Answer 460

- 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

Answer 461

Two - 1 extraceullar for LDL binding - 1 clathrin coat binding site

Answer 462

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

Answer 463

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

Answer 464

Golgi - COP1 | ER - COP11

Answer 465

Phagocytosis

Answer 466

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

Answer 467

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

Answer 468

Exocytosis is responsible for secretion of hormones, digestive enzymes, recycling of plasma membrane receptors and neuronal communication

Answer 469

Release of highly-concentrated insulin from pancreatic beta cells It happens only in response to high glucose

Answer 470

by researching exocytosis

Answer 471

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

Answer 472

SNAP25 contributes two Syntaxin contributes one VAMP contributes one

Answer 473

Attacks SNARE proteins

Answer 474

Botulinum - After consumption of contaminated food Tetnus - After skin cuts

Answer 475

- 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

Answer 476

Cosmetic - anti wrinkle Muscular spasms Dystonias

Answer 477

Membrane protein synthesis Protein tagging by sugars (glycosylation) Protein activation by trimming (proteolytic processing)

Answer 478

To protect themselves from harsh environment | and provide a tagging mechanism

Answer 479

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

Answer 480

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

Answer 481

A channel found in the ER membrane - move protein from ribosome to ER

Answer 482

- 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

Answer 483

It will become stuck in the membrane of the ER

Answer 484

Signal peptidase

Answer 485

- 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)

Answer 486

ER-resident enzymes carry KDEL sequence

Answer 487

- 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)

Answer 488

- 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

Answer 489

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

Answer 490

Genetically-modified pigs lacking alpha-galactose | make organ transplantation possible

Answer 491

Carbohydrates of Band 3 protein in erythrocytes determine human blood compatibility

Answer 492

Blood group is determined by a single terminal galactose residue O - lacks a terminal Gal A. - acetylated Gal B - normal Gal

Answer 493

Occurs prior to secretion - Preproinsulin is cleaved to proinsulin in the ER - Proinsulin is cleaved to insulin in the secretory vesicles

Answer 494

- 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

Answer 495

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.

Answer 496

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

Answer 497

Insulin is synthesised in beta cells of islets and is secreted into the blood stream

Answer 498

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

Answer 499

Disulphide bridges are important in giving structure to extracellular proteins

Answer 500

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

Answer 501

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

Answer 502

RAS pathway | RAS indépendant signalling pathway

Answer 503

- 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

Answer 504

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

Answer 505

- 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

Answer 506

Microarrays PCR Promotor bashing

Answer 507

To amplify DNA To clone DNA from minute samples To quantify levels of RNA

Answer 508

- 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

Answer 509

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

Answer 510

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

Answer 511

Can be used to investigate RNA which is specifically protein coding unlike DNA which has non-coding sequences (introns)

Answer 512

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

Answer 513

- 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

Answer 514

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

Answer 515

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

Answer 516

Tissue growth containing excessive numbers of cells

Answer 517

Tissue growth containing displacing but otherwise normal cells

Answer 518

Tissue growth where cells appear abnormal

Answer 519

Invasive, abnormal tissue growth | - Invasion of tissues

Answer 520

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

Answer 521

The more aggressive cancers tend to be further down the list of tumour types (neoplasm) suggesting a direction of travel as cancer cells emerge

Answer 522

It allows us to predict the common behaviours among diverse tumours despite radically different functions

Answer 523

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

Answer 524

Environmental

Answer 525

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

Answer 526

- Retinoblastoma - Wilms tumour - APC - Breast cancer

Answer 527

- 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

Answer 528

It is a straight line y = mX+c P(C1) ∝Age (they are proportional)

Answer 529

P(T)=P(C1).(PC2).(PC3)…..(PCN) x A^N Where N is the number of changes, and A is age

Answer 530

P(T)=P(C1).(PC2).(PC3)…..(PCN) x A^N equals LogP(T )= nLogA + constant This is the same is Y = mX + C

Answer 531

6 | The slope of the graph

Answer 532

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

Answer 533

- 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

Answer 534

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

Answer 535

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

Answer 536

Tubulin staining can be used to see what stage of mitosis cells are in as microtubules align the spindles at the midpoint of mitosis

Answer 537

Stain live embryos or add GFP to specific gene | Can see live how the cell cycle occurs

Answer 538

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

Answer 539

Fission yeast | Budding yeast

Answer 540

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

Answer 541

Budding yeast divide by growing buds. The morphology of these buds can be used to see where the yeast is in the cell cycle

Answer 542

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.

Answer 543

The mutation we have introduced creates a gene product that is functional at one temperature and not at others (protein loses its shape)

Answer 544

If the cell cycle is disrupted then cannot get large colonies of the mutants to investigate

Answer 545

If injected with progesterone, the frog will lay eggs at any time of the year Large eggs

Answer 546

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

Answer 547

There are specific cyclins and Cdks bound which target specific substrates relevant to a specific phase of the cell cycle

Answer 548

They increase and then are destroyed by proteolytic destruction

Answer 549

They infected cells at different stages in the cell cycle with Sendai virus, which then fused cells forming hetertropic fusions

Answer 550

- 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.

Answer 551

That there is something in mitotic cells which is dominant and capable of causing condensation of chromosomes anywhere in the cell cycle

Answer 552

- 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

Answer 553

There must be a factor that causes oocytes to enter metaphase - Maturating promoting factor (MPF).

Answer 554

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

Answer 555

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

Answer 556

Investigating temperature sensitive mutants, in this case Pombe yeast. These dividing yeasts were stained to see the nucleus and cleavage during mitosis

Answer 557

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

Answer 558

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)

Answer 559

Shared homology to a protein purified from frog eggs, showed it was evolutionary conservation

Answer 560

It would rescue the phenotype | - Shows evolutionary conservation

Answer 561

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

Answer 562

Tens of thousands in mammals | One in bacteria

Answer 563

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.

Answer 564

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

Answer 565

- 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

Answer 566

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

Answer 567

Antibodies that recognise the pre-replication complex and Cdt1 are profoundly useful for detecting malignant cells in cervical samples. E.g. smear tests

Answer 568

CDK regulators include activators, mainly the cyclins, and inhibitors, generically known as CKIs (CDK kinase inhibitors). Inhibitors mainly operate in G1 and S

BMS238 Cell and Molecular Biology Flashcards

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