Lectures (1-15) Imported Flashcards
The Rox1 gene contains a binding site for the Rox1 protein itself, what is the significance of this
Rox1 regulates its own transcription
What can be said about the orientation of the two polynucleotide chains in a dsDNA molecule
They are orientated antiparallel to each other
Give an example of a simple genetic switch
The tryptophan repressor protein represses genes required for tryptophan synthesis and storage. When levels of tryptophan are high then it turns off genes required for tryptophan synthesis
Pairing of homologues before segregation allows for crossing-over via homologous recombination, T or F
T
The human sliding clamp protein, proliferating cell nuclear antigen (PCNA) has a near identical structure to the homologue in yeast. What is PCNA significance in cancer
PCNA is a useful marker for hyperproliferative cells found in tumours
Explain the role of ribonuclease H in DNA replication
Ribonuclease H removes the RNA primers in the initial DNA-RNA hybrid molecule
Describe the structure of kinesin
The kinesin motor protein is a dimer that consists of two identical motor heads. Each head consists of a catalytic core and a neck linker
Describe the structure of a coiled-coil
Two ? helices wrap around eachother to form a stable structure. One side of each helix contains mostly aliphatic amino acids (such as leucine and valine). The other side contains mostly polar residues. The two helices are amphipathic and contain distinct hydrophobic and polar side chains. These two amphipathic helices align with hydrophobic residues packed tightly in the centre of the structure and polar hydrophilic faces exposed to the solvent.
Recall the pyrimidine bases
Thymine, cytosine, uracil
How many families of tRNAs are there
49
How does gene replacement work
Gene replacement is used to make small changes to endogenous genes in mice to see if these elicit diseased phenotypes
What change in molecular weight is seen as a result of phosphorylation
+95Da
Explain how the reverse transcriptase self-encoded by non-retroviral PolyA retrotransposons also acts as an endonuclease
As well as producing the new DNA copy of the RNA intermediate created from the non-retroviral retrotransposon, the RT self-encoded by it alsi creates a nick in the target DNA. This allows for the physical insertion of the newly synthesised DNA copy of the PolyA transcript into this cleavage site
Which proteins maintain the unwound parental DNA strands in a single stranded conformation and hence ease replication fork progression
SSBs – single stranded binding proteins
Explain how X-ray crystallography can be used to infer information about protein structure
A high energy focussed beam of electrons is fired through a protein crystal. Most of these x-rays pass straight through the crystal but some are deflected back. This gives rise to a diffraction pattern that is unique to each protein. The structure of the protein can then effectively be traced back to its diffraction pattern
During which stage of mitosis do the chromosomes attach to the mitotic spindle via their kinetochores and the microtubules
Metaphase
Binding sites for the same DNA binding protein all usually show a similar frequency of bases at the same location in binding site genes, T or F
T
What is the main function of circular dichroism
Used to induce secondary structure in proteins
Xeroderma pigmentosum is a disease caused by defective nucleotide excision repair machinery, what is the effect of this on patients
XP renders patients extremely sensitive to sunlight-induced skin cancer
What happens during G2 phase of the cell cycle
More growth of the cell and environmental checking. It has a shorter duration than G1 phase
Describe the role of rRNAs
Ribosomal RNAs are a major constituent of ribosomes. They are very large and very abundant and catalyse protein synthesis
The X-chromosome copy that is silenced is determined by maternal gene expression in the fertilised oocyte, T or F
F - Initial selection of the chromosome for silencing is random
What does SH2 stand for
Src homology 2 domain
Explain how the tools for a yeast-2-hybrid screen can be created
Separate the binding domain (BD) and activation domain (AD) of a regulatory protein that normally binds to the DNA to upregulate expression of a reporter gene. Fuse the protein of interest to the BD of the regulatory protein, now referred to as the bait. Fuse the AD of the regulatory protein with various peptide sequences from a cDNA library containing potential interacting peptides. These fusion proteins are referred to as the prey. Yeast cells are then cotransformed with BD and AD expression plasmids. Each transformant has the bait plasmid and one of the plasmids from the prey library.
The process of DNA transposon movement throughout the genome is also referred to as non-replicative transposition, T or F
T
How is DNA referred to that has been produced by ligation of multiple sequences from different sources
Recombinant DNA
What do SH2 domains bind to
Phosphorylated tyrosine residues
Describe the process of affinity chromatography
Cytosol is added to a column containing beads with a covalently attached substrate. Enzymes for the substrate that is bound to the bead will 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. The bound protein(s) can then be eluted from the column using a competing ligand to dislodge the affinity interaction. This allows separation of specific substrate binding proteins from the cytosol
What is meant by the processivity of DNA polymerases
Processivity refers the tendency of polymerases to continue to synthesise as long as there is sufficient substrate available
What can be said about the recombination of the construct into the mouse genome
The DNA repair mechanism machinery is not very efficient. Either the construct is not inserted into the mouse genome at all, or, non-homologous recombination occurs and the NEO and TK genes are inserted into the mouse genome. The TK gene is inserted into the construct to mark where non-homologous recombination has occurred
Explain how homologous recombination is mediated
Firstly, the 5’ ends are resected by exonuclease to create single strands that can be used to prime DNA synthesis when annealed to a template strand from the complimentary chromosome. The DNA-binding protein RecA promotes strand invasion of the undamaged template molecule by one strand from the damaged DNA molecule that acts as a primer. This forces complimentary binding with the same sequence in the undamaged sister chromatid. This forms a heteroduplex structure between the dsDNA helix of the sister chromatid and the single stranded sequence from the damage DNA molecule. This facilitates templated DNA synthesis of one strand by DNA polymerase. DNA polymerase synthesises across the damaged region by reading information out of the undamaged sister chromatid. The newly synthesised DNA then dissociates from its template and re-anneals to its original partner strand allowing second strand synthesis and formation of a pair of staggered singles stranded nicks. These nicks are then ligated by DNA ligase
Lysine acetylation is an indication of what
Transcriptionally active genes
What adapter molecule is required for translation
Transfer RNA (tRNA)
What is meant by high throughput
Small scale, fast and automated
What is meant when we refer to non-homologous end joining being quick and dirty
Non-homologous end joining is a far from optimal repair mechanism. It is carried out quickly but in itself, can induce mutations
What are the two constituent parts of each nuclear chromosome
Linear DNA molecule and proteins that confer specialised functions called chromatin
Describe the structure and interaction of leucine zipper domains with DNA
Leucine zippers consist of 2 ? helices that form a dimer held together by hydrophobic amino acids such as leucine. These domains straddle the DNA binding to symmetrical sequences in the case of homodimers, or non-identical sequences if the two helices are a heterodimer
Specifically what is the effect of histone acetylation on gene transcription
Acetylation of histones creates binding sites for transcriptional activation factors that contain a bromodomain. Histone Acetylation is associated primarily with transcriptionally active promoter sequences.
Describe the role of zinc finger domains
Zinc finger domains are found in the most frequency classes of transcription factors and are DNA binding domains that recognise 3-base pairs of double-stranded DNA
Recall the four hypothesised mechanisms of regulating transcription factors
Protein synthesis of inhibitors etc. Ligand binding, protein phosphorylation and addition of subunits
What is the result of some mutations in the src-tyrosine kinase
Some sarcoma cancers
What is the name of the enzyme that catalyses the addition of an amino acid to a tRNA molecule
Aminoacyl-tRNA synthetase
Creating homozygous knockout mice can take over a year, T or F
T
What is meant by the term transcription factor synergy
Transcription factors cooperate in order to influence gene transcription. Binding of one transcription factor to another may help prevent them from falling off the DNA. Instead of one interaction, each protein would need to lose two interactions to fall off the DNA. Similarly, binding of one transcription factor to DNA may enable another transcription factor to bind to that sequence also
Which ions bind to EF Hand domains in a structural or signalling mode
Ca2+ and Mg2+
Like proteins, DNA has unlimited topology and can adopt many shapes, T or F
F – DNA has a limited topology and defined 3D structures due to set interactions between the 4 bases following certain rules
What percentage of mammalian sperm and eggs contain extra chromosomes or missing homologues
4% of sperm, 20% of eggs
Polyacrylamide gel electrophoresis and Western blotting can also be used to determine protein interactions, T or F
T
Give example of helicase loading proteins that bind to ORC
Cdc6 and Cdt1
What can be said about the expression of molecular chaperones at high temperatures
Expression of hsps is elevated when the temperature is raised above normal. This is because high temperatures can cause properly folded proteins to become misfolded
What attribute of protein interactions can be determined using competitive ELISA
How strongly an antibody binds to the protein of interest
Temporal separation of replicator selection and origin activation ensures what
Each origin is used and each chromosome is only replicated once per cell cycle
How do proteins that increase polymerase processivity act
Sliding clamp proteins keep the DNA polymerase enzyme at the primer template junction by fixing itself to the primer template junction through association with a protein called a clamp loader.
Different sizes and valencies of metal ions are liganded by different numbers of amino acids and have different structural requisites, T or F
T
Other than GST, what other tag can be used in affinity chromatography to investigate protein interactions
Hexa-histidine (6xHis)
What happens at the metaphase to anaphase transition
Cell checks that all the chromosomes are attached to the mitotic spindle before triggering anaphase and completing cell division
What is meant by the DNA code being non-overlapping
One triplet/codon is read at a time, followed by the next three bases (i.e. CGATTG –> CGA + TTG, CGATTG –> CGA + GAT TGX…)
Explain how electrophoretic mobility shift assays can be used to identify DNA binding proteins
EMSA involves the radioactive labelling of a known sequence of DNA that contains the binding site to which you want to identify interacting proteins for. This labelled DNA in then mixed with purified proteins or cell extracts. Instead of adding DNAse and heating, the mixture is then immediately run by gel electrophoresis. If a protein has bound to the labelled sequence, then it won’t move as far though the gel as the unbound DNA and would be represented by an additional band in the gel
How do aromatic amino acids interact with water
They don’t, aromatic amino acids are hydrophobic
Explain how X-chromosome inactivation accounts for tortoiseshell or calico cats
Calico cats are exclusively female. They are heterozygous for two coat pigment alleles, black and orange. Early in development in progenitor cells either orange or black alleles are inactivated. Where black pigment allele containing X-chromosomes are switched off, all progenitors will produce fur orange in colour. Where orange pigment allele containing X-chromosomes are switched off, all progenitors will produce fur black in colour.
Which enzyme joins 3’ and 5’ ends of the Okazaki fragments together
DNA ligase
Which motor protein moves vesicles and other organelles towards the +end of the microtubules
Kinesin
Where in the kinesin structure does the organelle attach to
The other end of the long coiled-coil that holds the two motor heads together
What is the purpose of regulating the rate of division in cells and tissue
Enables you to maintain cell numbers in each tissue
What is the initial product of DNA replication
A DNA-RNA hybrid molecule
Binding of SH2 domains to their phosphotyrosine ligands is involved in the formation of signalling complexes, T or F
T
Describe chemically, how amino acids are added to the 3’ end of the tRNA
Ester bond forms between the carboxyl group of the amino acid and the ribose group of the last nucleotide
Genes can be on either the sense or antisense stands of the DNA but not both, T or F
T
Recall the general and word equation for the first reaction in DNA ligase activity
ATP + 5’P –> PPi + 5’P-AMP. Adenosine trisphosphate + 5’ phosphate –> pyrophosphate + 5’adenosine diphosphate
RNA primers are required throughout both the leading and lagging strand synthesis, T or F
F – they are only required at the start of lagging strand synthesis but throughout leading strand synthesis
What are the 4 different ways of representing protein structure
Sticks, space-filling, ribbon and backbone
Explain how to construct a radioactive binding curve to investigate protein interactions
Attach the protein of interest to beads by incubating the beads with an excess of the protein ligand. Unbound protein ligand can then be separated using centrifugation. Increasing the amount of ligand will increase the amount of ligand binding to the protein of interest leading to saturation of the binding sites. You can then record the amount of ligand bound per binding site and plot this against the concentration of ligand added.
What happens as a result of selective removal of linker histone H1 from the core nucleosome
Causes de-condensation of the chromatin during interphase that allows gene transcription
How did Conrad Waddington describe how cells differentiate due to epigenetic changes
Cells traverse and epigenetic landscape of various differentiation possibility which gradually restricts cell fates
What is the enzyme responsible for the ubiquitination of lysine residues
Ubiquitin ligase
Which amino acid residues form hydrogen bonds with water molecules and are exposed on the outside of the proteins
Hydrophilic polar side chains
What causes the synthesis of each Okazaki fragment in the lagging strand to halt
When DNA polymerase reaches the RNA primer attached to the 5’ end of another fragment
Explain how fluorescent resonance energy transfer can be used to investigate protein interactions
Recombinant fusion of protein X and Y to separate fluorescent proteins that absorb and emit certain wavelengths of light allows you to determine if X and Y interact/bind. By correlating the wavelength emitted by the fluorescent protein attached to X with the wavelength of light needed for fluorescence of protein Y you can activate protein Y fluorescence if it is in close proximity to X (i.e. it is bound). I.e. if shining light needed for fluorescence in protein X leads to the appearance of light that is given off as a result of protein Y fluorescent you can determine that X and Y interact
Describe the composition of the core nucleosomes which the DNA sequence wraps around
Core nucleosomes are proteins that consist of 8 distinct subunits called histones. The DNA sequence wraps twice around each core nucleosome
Ligation of newly synthesised adjacent DNA fragments is a two-step reaction, requiring ATP hydrolysis, T or F
T
Non-retroviral retrotransposons have expanded hugely in numbers during evolution of higher mammal genomes, T or F
T
How often do hydrogen bonds occur in the primary sequence of an ? helix
Every 3.6 residues
What bonds are formed during the process of DNA replication
Phosphodiester bonds
During which phase of mitosis do the sister chromatids condense
Prophase
What happens during anaphase
Sister chromatids are separated
Which type of transposons make up the majority of transposons
Retroviral retrotransposons
Transcriptional activators recruit ATP-dependant chromatin remodelling enzymes, what four ways can these enzymes act to upregulate gene transcription
Selective histone octamer and whole nucleosome remodelling, selective histone removal and replacement and/or by recruiting code writers and readers
Random coils are another secondary structure of proteins and can connect ? sheets together, T or F
T
What are the stage of mitosis
Prophase, prometaphase, metaphase, anaphase and telophase
What three things are important when creating cDNA libraries from mRNA
Only one cDNA insert is inserted into each plasmid, this is done by controlling concentrations. Once plasmid only must be taken up into each bacterium and one bacteria must start each colony.
What is the role of SH2
Acts as an important phosphotyrosine binding domain that is often involved in signalling mechanisms
What attribute of enhancer sequences results in the need of insulators and barriers
Enhancers are binding sites for transcriptional activators. These sequences are usually promiscuous and activate transcription of any adjacent genes. Insulators and barriers are required
What is significant about the N-terminals of core histones
These project out from the nucleosome core and are free to interact with other proteins. These tails are rich in lysine residues and facilitate regulation of chromatin structure and function. They interact with proteins that effect the ability of the chromatin to be de-condensed, re-condensed and transcribed
Explain the process of non-homologous end joining
The ends of the double stranded break are rendered flush with loss of bases via degradation from the ends of the strands. These flush ends are then ligated together. However, this leads to a loss of DNA sequence due to the degradation that occurs prior to ligation.
Each de-condensed chromosome occupies a specific region in the interphase nuclei, explain this phenomenon
As genes are transcribed the relative position of the chromosome in the nucleus changes. At interphase transcriptionally inactive regions of/chromosomes become localised at the periphery of the nucleus. In contrast, transcriptional activation of a gene is accompanied by movement of the gene towards the centre of the nucleus
What is significant about the function of the XP genes
Their function is tightly coupled and targeted to regions of the genome that are highly transcribed. This acts as a surveillance system to signify regions of the genome that are transcribed. RNA polymerase required for transcription is physically coupled to the DNA repair machinery
Give some examples of aromatic amino acids
Tyrosine, proline
What is the difference in the structures of the two kinase domains of src-tyrosine kinase
The large kinase domain is mostly ? helical in structure whereas the smaller kinase domain is mostly a ? sheet
What are the two purposes of homologous recombination between non-sister chromatids
Aligns the chromosomes ready for anaphase and facilitates formation of the synaptonemal complex as well as allowing for genetic recombination between paternal and maternal DNA on the same chromosome
Define what is meant by a kinetochore
Protein complex that binds to the microtubules in the mitotic spindle
How do specific or regulatory transcription factors differ from permissive transcription factors
Specific or regulatory transcription factors bind to specific genes are play a regulatory role in transcription. They can be transcriptional activators or transcriptional repressors and bind anywhere in the gene, sometimes quite far away.
What is the name given to the sequence in the DNA to which transcriptional repressors bind
Silencers
Describe the processes that occur during translation termination, after a stop codon is reached
Stop codons aren’t recognised by a tRNA molecule and thus don’t code for a corresponding amino acid. Once a stop codon is present in the A site of the ribosome, protein release factors bind to the site and terminate the polypeptide chain. Peptidyl transferase then catalyses the transfer of H2O to the C-terminus of the polypeptide chain resulting in the formation of a carbonyl group (COOH) and release of the protein from the ribosome. Release factors then move into the P site causing the ribosomal subunits to dissociate
Give examples of different cyclins and how they act with Cdks at different times in the cell cycle
M-cyclin is high during mitosis and the Cdk-M-cyclin complex leads to phosphorylation of protein involved in the assembly of the mitosis machinery. Whereas S-cyclin is high during S phase and the Cdk-S-cyclin complex phosphorylates proteins involved in the assembly of DNA replication machinery
Explain how a genomic library is created
The whole genome of the organism is cut into fragments and each fragment is cloned into a different plasmid vector to create colonies with each fragment sequence
Describe an ? helical structure
Spiral conformation in which every backbone amino group donates a hydrogen bond to the backbone carboxyl group of the amino acid located 3-4 residues earlier in the protein sequence
What happens when cytosine is deaminated and what are the downstream effects of this
Converted to uracil. This will still pair with guanine but during replication the guanine will be replaced with an adenine, leading to a nucleotide substitution from CG–>TA
What is meant by the quaternary structure of a protein
The relationship between individual proteins in a multimeric complex
What chemical mutagen is commonly used in forward genetic screens
EMS
General transcription factors are required for all gene transcription, how do they act
They act to guide RNA polymerase and bind to the promoter sequence
How do retrotransposons act in a similar way to retroviruses
They replicate via RNA intermediates and produce new DNA copies that integrate at new genomic locations. Retrotransposons self-encode the reverse transcriptase enzyme required for this mechanism
Alu and B1 are human non-retroviral retrotransposons, explain what is significant about these two transposable DNA sequences
They evolved from the same sequence element – 90million years ago. Alu and B1 originate from a single copy of the 7SL RNA gene
Explain how the chromatin is indirectly linked to the microtubules during chromosome segregation
The CENP-A histone containing chromatin physically binds to the kinetochore inner plate. An interaction between the kinetochore inner and outer plates links this chromatin to the microtubules of the mitotic spindle
Restriction enzymes have precise recognition sites, T or F
T
Describe the basic structure of an EF-Hand domain
Consists of two ? helices linked by a short loop region of around 12 amino acids that bind to Ca2+
The primer sequence of DNA synthesis can only grow in a 3’ to 5’ direction, why is this
The addition of a dNTP can only occur by the nucleophilic attack of the 3’ carbon and hydroxyl on the 5’ phosphate of the incoming nucleotide
How many different human codons are there
61
What factors recognise stop codons and trigger dissociation of the ribosomal subunits
Release factors – molecular mimics that enter the A-site and cause dissociation
What examples of simple systems are used in forward genetic screens
Drosophila, C. elegans, Zebrafish and Yeast
Surface plasmon resonance cannot be used to image protein interactions in real-time, T or F
F – it can, huge advantage
The price of human genome sequencing has decreased from $100m to $8k today, T or F
T
Housekeeping genes are often cloned more often when creating cDNA libraries, why is this
They are highly transcribed
What is meant by the consensus sequence
The consensus sequence shows the generally conserved sequence common to all binding sites for a DNA binding protein. This can be used to identify other binding sites with the same consensus sequence and that may bind to a DNA binding protein of interest.
Describe the localisation of the riboproteins and protein synthesising regions of the ribosome within its structure
Riboproteins are found on the surface of ribosomes whilst the protein synthesising regions are deep within the structure
What is the name given to specific DNA sequences that direct the initiation of DNA replication by recruiting replication initiation proteins
Replicator sequences
Of what order can the telomeric repeat sequences at the ends of chromosomes be
Hundreds
What is the other term used to describe aminoacyl tRNAs
Charged tRNA
What method other than Edman degradation (chemical disruption) can be used to infer primary structure of a protein
Physical disruption through mass spectrometry
What type of bonding holds DNA binding proteins in place at the major groove of the DNA
hydrogen bonds
To identify genes that affect a specific process, thousands of F2 families need to be screened, T or F
T
What is meant by the P site of the ribosome
Peptidyl tRNA site
The more ordered and symmetrical the protein specimen is that is used in cryo-EM, the easier the averaging process, T or F
T
How are genetic distances calculated in humans
A correlation between known SNPs and diseased phenotypes are investigated. SNPs act as the markers and over 1m have been identified in humans. The offspring of a diseased individual are analysed to determine if particularly SNPs are always present in the diseased state. If an SNP is always present in diseased offspring but not healthy ones you can determine that the mutant gene is linked to that SNP. You can then correlate the location of the known SNP with known genes to derived candidate genes.
What are the products of genes encoded by the genes mutated in xeroderma pigmentosum
These genes encode proteins that participate in the nucleotide excision repair pathway
Other than epigenetics and in histones, what other role can acetylation play
Acetylation of tubulin stabilises the microtubules
Phosphatases and kinases used PH domains and have a direct role in lipid signalling, T or F
F – kinases and phospholipases contain PH domains involved in lipid signalling
Give an example of a disease caused by a misfolded protein
Creutzfeldt–Jakob disease is caused by misfolded pathogenic proteins knowns as prions that enter the brain and convert normal proteins into misfolded ones. This seeds new cross-? filaments of protein aggregates.
Which amino acid residues have high helix forming propensities
Methionine, alanine, leucine, lysine and glutamate
How are cDNA libraries created
Firstly, mRNA is extracted from a cell. Reverse transcriptase then coverts this single stranded mRNA to dsDNA – referred to as cDNA. The cDNA from each mRNA is then cloned and undergoes ligation and transformation to get each sequence into bacteria.
How is transformation of bacteria achieved once recombination of a plasmid vector has occurred
The bacteria are missed with the recombinant plasmid vector and their membranes are permeabilised by electroporation or with chemical treatment. Competent bacteria will take up the new DNA
SH3 recognises proline-rich motifs, how does aromatic stacking account for the interdigitating of tyrosine residues with the target poly-proline domains
Aromatic stacking is the process by which the tyrosines contained within the SH3 domain interdigitate with proline residues within the binding proteins target domain. This is caused by an interaction between the aromatic side chains of tyrosine and proline residues with hydrogen atoms attached to the adjacent aromatic ring. This interaction is a dipole-dipole interaction due to the ?- aromatic rings and the ?+ hydrogen atoms. The aromatic residues (tyrosine) of the SH3 domain are positioned so that they can stack with aromatic (proline) residues contained in the proline-rich motifs
Explain how the Sanger sequencing method has been automated
Rather than radioactively labelling primers, the ddNTPs used in sequencing are tagged with fluorescent dyes, eye base with a different colour. Then the dsDNA template strand is denatured by heating to break the hydrogen bonds between complimentary base pairs. DNA polymerase and a batch of primers are then added to the mixture and DNA synthesis can occur. At random points in the newly synthesised DNA strand a ddNTP will be incorporated into the strand resulting in termination of DNA synthesis. This will produce DNA strands with ddNTPs at every position in the sequence. These strands can then be separated on a gel allowing the sequences to flow continuously. A camera then measures the fluorescence at a fixed point in the gel as the DNA sequences flow underneath. This camera records the colour of the fluorescence corresponding to each base at each position in the sequence. The results are then presented as a graph showing the intensity of fluorescence over time, this is known as a trace and allows the computer to determine the DNA sequence base-by-base.
Recall the four logic-based mechanisms by which transcription factors can regulate the transcription of other transcription factors
Positive feedback, negative feedback, flip-flop devices and feedforward mechanisms
What technique allows you to identify the specific gene mutated in a particular phenotype
Positional cloning/linkage analysis
Give some examples of diseases associated with protein misfolding
Huntington’s, cystic fibrosis (CFTR), cancer (p53) and Alzheimer’s
What is the role of the start checkpoint
Checkpoint at the end of G1 that checks to see if the environment is favourable before triggering the DNA replication machinery to replicate the DNA. It ensures that the cells have enough resources to go through the cell cycle to G1 again
What are the four main types of DNA damage
Deamination, depuration, pyrimidine dimers, DNA breaks
Give an example of a specialised histone that mediates the attachment of the chromosome to the kinetochore inner plate
CENP-A
Which method was used to sequence the human genome
A combination of shotgun and progressive sequencing
Which enzyme is responsible for the synthesis of new DNA daughter strands
DNA polymerase
What post-translational modification targets proteins for transport to the nucleus of the cell
SUMOylation
Transcription factors recognise short stretches of DNA through interactions with individual base pairs, T or F
T
Why are regions on the face, neck, bely and white still white in calico cats, despite X-chromosome inactivation
These regions don’t contain pigment cells
What enzyme is responsible for the synthesis of RNA primers
DNA primase
Telomeres define chromosome ends and maintain chromosome integrity, T or F
T
What is meant by the genetic code being degenerate
Some amino acid acids are specified by more than one different codon
Which end of the tRNA strand contains the bound amino acid
The 3’ end
Explain the main differences between equilibrium and velocity density centrifugation
Velocity based density centrifugation involves the extraction of the different density fractions based on how long it takes them to deposit at the bottom of the tube whereas equilibrium based density centrifugation relies on prolonged high speed centrifugation to separate the different density organelles within the steep sucrose gradient.
Werner’s syndrome is an example of a disease caused by mutations in a helicase. Describe the aetiology and symptoms of this condition
Werner’s syndrome is a progeria (premature ageing) caused by a autosomal recessive mutation (loss of function) in the RECQ helicase encoded by the WRN gene
How is the end replication problem overcome
Addition of non-coding telomeric repeat sequences to the 3’ end of the DNA sequence. These are long enough to enable DNA primase to bind and initiate new RNA primer synthesis and prevent chromosome shortening
Outline the typical methods used in protein purification
Intitially you start with ion exchange chromatography which acts as a rough separation method based on charge. The fractions produced from ion exchange chromatography are then used in gel filtration chromatography to separate the fractions further, based on size. In the final stage, these fractions are then separated further using affinity chromatography. A combination of two or three different chromatography techniques is used to separate pure proteins.
Describe how the process of translation is initiated
Initiator tRNA carrying methionine is loaded into the small ribosomal subunit with eIF-2. Met-charged tRNA is the only aminoacyl tRNA molecule capable of binding directly to the small ribosomal subunit and the only charged tRNA that can bind directly to the P site of the ribosome leading the A site vacant. Whilst the met-charged tRNA binds to the large ribosomal subunit, the small ribosomal subunit binds to the capped 5’ end of the mRNA and begins progressing along the strand until the met start codon AUG is reached. Once this AUG is reached the eIF’s dissociate and the large ribosomal subunit fully assembles
In order for sticky end ligation to occur from restriction fragments, the restriction enzymes need to have identical recognition sites, T or F
F – as long as the sticky ends have cohesive overhangs i.e. complimentary bases they can ligate with or without identical recognition sites
Explain the different functions of zinc finger domains in different proteins
Zinc fingers perform a structural function in protein-DNA interactions or a catalytic function in proteins such as botulinum toxin
What is significant and unusual about DNA binding proteins that allows them to bind to DNA
DNA binding proteins have a positive charge due to high positively charged amino acids contained within them. This allows the protein to interact and remain bound to the negatively charge phosphate backbone
What is meant by the minimum consensus sequence for SH3
The smallest region which SH3 will bind to. This is the P-x-x-P motif consisting of a 4 residue sequence with first and last residues being prolines
Multiple ribosomes can bind to the same mRNA, T or F
T – this is referred to as a polysome
How are tRNAs with attached amino acids referred to
Aminoacyl-tRNAs or charged tRNAs
What are the key differences between RNA and DNA
RNA replaces thymine with uracil. RNA is also synthesised as a single strand and thus is unstable and rapidly degraded. Finally RNA contains a ribose sugar backbone instead of a deoxyribose sugar. The difference is an -OH group replaces the H bonded to the 2’ carbon in the sugar ring
Which residue does enhancer of zeste act on and what type of modification is it
Enhancer of zeste methylates lysine 27
Which metal ions bind to their binding domain and have a structural role, give some examples of protein that contain these domains
Zn2+ ions bind to zinc domains. Examples of proteins with these domains include botulinum toxin, zinc fingers and DNA binding proteins
What are alpha-satellite DNA repeats that are found within centromeres
Alpha-satellite DNA repeat sequences are repeat sequence elements around 170bps that act as specific binding sites for a set of specialised histones localised to centrometic sequences. These act as target interaction proteins for kinetochores
Plasmid vectors can only hold up to 30kbps of DNA, what vector is used for DNA fragments larger than this
Bacterial artificial chromosome (BAC) which can hold up to 300kbps
An incoming nucleotide can only be added to the free 5’ hydroxyl group on the terminal deoxyribose sugar of an existing polynucleotide chain, T or F
F - incoming nucleotides can only be added to the free 3’ hydroxyl group
How is transcription different from DNA replication
Multiple RNA polymerases bind to the same gene, no primers are needed, only one strand of DNA is used as the template and polymerase only moves in one direction. Finally the transcript doesn’t remain bound to the template as in semi-conservative replication
What chemical modifications can lead to DNA damage
Hydrolysis, oxidation or random uncontrolled methylation
Describe the process of dideoxy terminator/chain termination/Sanger sequencing
Firstly, you start with a dsDNA sequence of interest and this is denatured by heating to 100?C to break the hydrogen bonds between complimentary base pairs and leave ssDNA templates. The template strand is then allowed to cool in the presence of radioactively labelled primers allowing them to anneal. DNA synthesis is then allowed to occur by adding DNA polymerase and deoxynucleotide trisphosphates. A mix of dideoxy nucleotide trisphosphates are also added into the mix, that prevent the subsequent synthesis of DNA once they have been incorporated into the growing polynucleotide strand. By having a mix of deoxy and dideoxy nucleotides, different strands will end at different positions. These strands can then be separated on a gel to produce distinct bands based on template strands that have terminated at each position. Running all four ddNTP reactions on the same gel results in a nucleotide ladder which allows for sequencing base by base.
Explain the role of linker histones such as H1
Linker histones act as straps that connect the incoming and outgoing strands of DNA that wrap around the core nucleosome. This helps to stabilise the formation of the 30nm chromatin fibres
Give an example of a promoter sequence to which a general transcription factor binds to
TATA box – consisting of a TATAA/TAA/T sequence that lies 30 base pairs upstream of the coding sequence
What defines the working range of the resin in gel filtration (size exclusion) chromatography
The sieving effect of the beads due to the pore size. Proteins too large are excluded from moving through the column
What is meant by a sequence logo
A sequence logo is a size representation of the bases within a DNA sequence where the size of the letters represents the frequency of bases within the sequence
Histone methyltransferases can modify many different lysine or arginine residues, T or F
F – histone methyltransferase exhibit exquisite site specificities
Methylation of core histones creates binding sites for transcriptional repressors that contain what kind of domain
Bromodomain
As well as histones, DNA can also be modified by methylation. When and how does this occur
There is a close functional relationship exists between transcriptionally repressive histone methylation and corresponding DNA methylation on cytosine bases. The addition of methyl groups to cytosine residues is mediated by DNA methyltransferases (DNMTs). Transcriptionally inactive promoters are frequently rich in methylated CpG dinucleotides
Give an example of another helicase mutation that causes disease, other than Werner’s syndrome
Bloom syndrome is another loss of function mutation that occurs in another Rec-Q family DNA helicase. The role of this helicase is to maintain genome integrity. This mutation causes a rare cancer phenotype with tumours in multiple tissues
Where/when is 2D gel electrophoresis commonly used
Proteomics and in complex samples
Which specialised sequence within chromosomes is the DNA sequence at which DNA replication is initiated
Replication origin
What do Y, H and N represent in the consensus sequences
Y represents either a C or a T. H represents and A, C or T. N refers to any base
What is meant by G1 phase and what is going on the cell during this phase
G1 or gap 1 phase is the phase in which most cells are in. The cell is growing and constantly checking its environment. This growth is required to maintain cell size with subsequent divisions.
Explain how microarray grids are made
The grids are made by a precise robot that places a spot on the array for each gene in the genome. Each spot contains one single stranded cDNA antisense strand for the gene. These are attached to a specially treated microscope slide so the ssDNA will stick to it.
How do regulatory transcription factors function
They interact with the RNA polymerase complex and either alter acetylation of the DNA (which effects chromatin structure), bind to other transcription factors or act upstream of permissive/general transcription factors
Monoubiquitination marks inappropriately folded proteins for degradation by the proteasome, T or F
F – this is the result of polyubiquitination
Which metal ion binding domains have a regulatory role and give some examples of proteins that contain these domains
Ca2+ binding domains have a regulatory role. These include calmodulin and synaptogenin
Pyrimidine dimers can only occur between identical adjacent pyrimidine bases, T or F
F – it can be the same pyrimidine or different ones (i.e. T-C, T-T, C-C)
Recall the purine bases
Adenine, guanine
Which sequence is perfectly conserved across all Rox1 binding sites
GTT
What does the prediction of the secondary structure rely on in order to achieve desired structures
The tendency of particular amino acids to form particular structures
Which proteins are required for vesicular and organelle transport along the microtubules
Dynein and kinesin
What maker is often used in fruit fly screens
Notch
How are separated DNA restriction fragments visualised after separation
Dyes such as ethidium bromide are added which stains the DNA when exposed to UV light
Histone methyltransferases act as regulators of gene transcription and are uniquely site specific, T or F
T
At which terminus does protein synthesis start at
Amino-terminus
Splicing is specific to eukaryotic transcription, T or F
T
Describe the ternary structure formed by proteins that increase processivity and how this complex acts
Sliding clamp positioning is ATP-dependant. A ternary structure is formed by the sliding clamp and clamp loader proteins and the associated ATP. This complex sits behind the DNA polymerase and provides an extra impetus to drive it forward
The organised representation of all of the chromosomes in a eukaryote at metaphase is canned the karyotype, T or F
T
Mistakes during meiosis I result in gametes with an extra chromosome or lacking a homologue, what is the name given to this event
Nondisjunction
What is meant by histone code writers
Histone methyltransferases are histone code writers, they act as an additional code on top of the genetic code i.e. epigenetic to
What is significant about viruses when it comes to protein synthesis
Viruses break the central dogma of DNA–>RNA–>Protein whereby reverse transcriptase enzymes can make DNA from RNA
Correct folding is a multistep process that must occur in the correct order. What is the effect of an incorrect or out of sequence step
May reduce the energy state of the protein but blocks further folding and may lead to a dead end
Explain the process of depurination
The carbon to nitrogen bond between the carbon position one in the deoxyribose sugar and the nitrogen in the purine ring is hydrolysed. This releases the base and results in the loss of a base pair, nucleotide deletion, in one of the daughter DNA molecules produced during replication
Explain what is meant by X-chromosome inactivation
X-chromosome inactivation is a process that occurs in mammals and acts as a dose compensation mechanism that equalizes the levels of X-chromosome derived gene products in males and females. One X chromosome copy is silenced in each somatic cell during early development of female embryo
What would be seen in the binding curve of a protein ligand that interacts more strongly with its target
Its binding curve will reach a maximum and plateau quicker as less ligand will be required for 50% binding saturation
EF-Hand motifs bind cations via 5 oxygen containing amino acid side chains, T or F
T
Define the tertiary structure of a protein
The way in which individual secondary structural elements pack together within a protein and between sub-domains of proteins
How can proteins that interact with DNA be identified
Attach a specific DNA sequence of interest to beads contained within the column. Pass a nuclear extract through the column and proteins that bind to this specific DNA sequence will be retained in the column. Unbound protein can be removed by washing the column through with a buffer. Finally bound proteins can be eluted from the DNA using high salt concentrations
What attribute of restriction enzymes accounts for their binding to palindromic recognition sites
Restriction enzymes bind as dimers
What do PH domains bind to
Phosphoinositide lipids
What is meant by the term secondary protein structure
Local folding of the primary amino acid sequence into three different types of structures; ? helices, ? sheets and random coils
Which type of transcription factors will bind to methylated lysine 9 and 27 residues
Transcriptional repressors containing bromodomains
Describe the insulin signalling pathway from ligand binding to recruitment of a scaffold protein, include reference to specific binding domains that are involved
Initially the insulin hormone binds to its receptor on the cell membrane. The activated receptor then autophosphorylates itself at tyrosine residues. Phosphotyrosine then recruits the insulin receptor substrate-1 (IRS1) protein via its PTB domain. The PH domain of IRS1 then binds to phosphoinositide lipids in the plasma membrane. The activated insulin receptor then also phosphorylates the now bound IRS1 at tyrosine residues. Phosphotyrosine within the IRS1 protein then binds to the SH2 domain of the adapter protein Grb2. Grb2 contains 2 SH3 domains and 1 SH2 domain. Once bound by its SH2 domain to IRS1, one of the SH3 domains binds to a proline-rich region of another protein called Sos. Sos then also binds to phosphoinositides in the plasma membrane via its PH domain. The other SH3 domain of Grb2 binds to a proline-rich sequence contained in a scaffold protein. Once the scaffold protein is bound it too binds several other signalling proteins which relay the signal further
CD gives a relative % or fraction of each of the secondary structure types in any protein by its unique spectrum, T or F
T
Explain how tags are used in immunoprecipitation to identify interacting proteins
HA, Myc or Flag peptides are fused to the protein of interest. The cytosol containing potential interacting partners and the fusion protein is added to a column containing beads covered in a bacterial protein that binds strongly to antibodies. In addition, anti-HA, anti-Myc or anti-Flag antibodies are also added to the column. These antibodies will bind to the target protein, together with any interacting proteins and then will bind via their Fc domain to the bacterial protein on the beads. Once the protein of interest and interacting proteins are bound via antibody interactions to the beads they can be analysed by Western Blotting or Mass Spectrometry
Describe how microarrays work
Firstly, the mRNA from a tissue is extracted and purified then tagged with a fluorescent dye. This tagged mRNA is then introduced onto the array and allowed to hybridise to the antisense cDNA in each spot. Only genes being expressed by the tissue will be transcribed and hence present in the mRNA. Thus, these will be the genes to hybridise to their respective cell in the array. A reader then uses a sensitive camera to detect which genes are on/transcribed in the tissue by measuring the fluorescence at each position in the grid. The grid coordinates that exhibit fluorescence can be used to determine the identity of the genes being expressed
Describe the structure and interaction of zinc finger domains with the DNA
Zinc finger domains consist of an ?-helix and B-sheet. The ?-helix interacts with the major groove of the DNA by interactions of arginine and histidine residues with bases in the DNA sequence. These domains require the presence of a Zn2+ ion to stabilise the structure and hold the domain in place. These domains are usually found in combination with several other zinc fingers
What is meant by SnRNPs and what do they consist of
Small nuclear ribonucleo proteins are structures that make up the spliceosome apparatus. They consist of small nuclear RNAs and proteins
What happens to the original site from which the DNA transposon was initially excised from
It is repaired by DNA repair mechanisms
What is the key difference between genetic and epigenetic modifications
Genetic alterations occur to the DNA sequence directly and permanently affect gene expression. Epigenetic alterations occur to chromatin structure and act to modulate gene expression. These do not alter the DNA sequence and are reversible
What is meant by DNA looping
Chromatin is quite stiff and so does not bend easily. It is thus thought that for two proteins to interact with the DNA they need to bind directly to neighbouring DNA sequences. These binding sequences for regulatory transcription factors need to be over 500 base pairs apart, this leads to DNA looping
Which types of amino acids residues are found in the middle of the ? strands/sheets and give examples
Aromatic residues such as tyrosine, phenylalanine, tryptophan, valine and isoleucine
Explain the role of enhancer of zeste in hox gene repression and the involvement of polycomb repressive complexes
Enhancer of zeste is the catalytic subunit that acts as part of a complex that represses hox gene expression. PRCs contains chromodomains and methylate lysine 27 loci toproduce global derepression of hox genes
What protein is said to enhance the processivity of polymerases
Sliding clamp proteins
How are DNA cloning vectors made
Plasmid vectors are made from plasmids by adding a series of restriction enzyme sites in one part of a plasmid called the multiple cloning site
What is the name of the complex that marks cyclins for degradation and how is this achieved
Anaphase promoting complex (APC) marks cyclins for degradation by ubiquitination
Why are more simple organisms used preferentially over more complex mammalian systems in forward genetic screens
Because the mutagenesis process occurs at random and effects the entire genome the probability if hitting a specific gene is relatively low. This increases the number of mutagenized animals you would need to analyse to find the interesting phenotypes. Thus simpler organisms are used to speed up this process
What two types or arrangements of ? strands in ? sheets can be observed
Parallel and antiparallel
Extensions of the two leucine zipper helices straddle the minor groove of the DNA, T or F
F – they straddle the major groove
What is meant by the E site of the ribosome
Ejection/empty site
Retrotransposons also move throughout the genome/chromosomes, T or F
F – retrotransposons never move. They act through RT converting RNA back to dsDNA at random points in the genome
What is the other product formed from the addition of a nucleotide to the growing polynucleotide chain
Pyrophosphate
How many different genes have been identified as being responsible for xeroderma pigmentosum, give some examples
7 different genes including XPA, XPC, XPD, XPF and XPG
How can DNA footprinting be used to confirm DNA binding proteins
Firstly, a DNA sequence is radioactively labelled. This DNA sequence is then subjected to hydrolysis which produces all possible DNA sequence lengths with an ever diminishing molecular weight. However, some size fragments will be excluded from the fragment profile due to masking by the bound radiolabel. These specific fragments are unique to each DNA sequence and will be represented by a gap in the DNA binding profile. This acts as a fingerprint and allows the identification of the sequence.
During which specific stage of the cell cycle can chromosomes easily be distinguished
Metaphase of mitosis
Explain how a forward genetic screen is carried out in flies
Male flies are exposed to a mutagen such as EMS which is then integrated into the body and introduces mutations in the sperm. This generation of males, referred to a P0 are then outcrossed with non-mutagenised wild type female flies. The male offspring from this cross are then collected, these are referred to as the F1 generation. Each male in the F1 will have 4 or 5 mutated genes. These males are then crossed with wild type females once again to produce the F2 generation. The F2 generation will contain both males and females with mutant copies of the genes. This family is then incrossed to see what the homozygous mutants look like.
What is meant by the secondary structure of RNAs
Secondary RNA structure refers to the base pairing that occurs within a single RNA strand.
What does SH3 stand for
Src homology 3 domain
What DNA replication event occurs in S phase
Origin activation – the unwinding of DNA and recruitment of DNA polymerase
Cdks require cyclin to become active, T or F
T
How is cyclin-dependant kinase (cdk) activity important in limiting pre-RC formation and activation to specific points in the cell cycle
Cdk levels are high during S phase of the cell cycle. High cdk levels leads to the phosphorylation of already formed pre-RC thus activating them and leading to formation of the replication origin. Cdk also acts to phosphorylate the individual components of the pre-RC, particularly the Cdc6, Cdt1 and ORC elements. Phosphorylation of these constituent elements leads to their inactivation and hence inhibition of new pre-RC formation during S phase. During G1 cdk levels are low and thus there is little phosphorylation of Cdc6 and Cdt1 and hence more pre-RC formation
Recall the carbon nomenclature for a deoxyribose sugar
Carbon 1 (1’) is the carbon to the right of the oxygen atom in the deoxyribose ring. Move round in a clockwise direction so that carbon 5 (5’) is the CH2OH group attached to the deoxyribose ring
Explain the process of velocity sedimentation type density centrifugation
A test tube containing a stabilising gradual sucrose gradient is established whereby highest sucrose concentrations are found at the bottom of the tube. Addition and centrifugation of the cytosol separates the organelles based on density. Heavier organelles will sediment quicker and lighter ones will take longer. Organelles deposit at the sucrose concentration that equates to their own 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.
The poly-acrylamide gel has a sieving effect and protein migration through the gel in the presence of SDS is proportional to molecular mass, T or F
T
Only the Met-tRNA with eIF-4A can bind to the P site of the small ribosomal subunit alone, T or F
F – met-tRNA is the only aminoacyl tRNA with eIF-2 bound that can bind the small ribosomal subunit alone
What features can you derive from the binding curve of ligand concentration against binding
Max binding stoichiometry - how much of the interacting protein can bind to the protein of interest. Association constant – affinity of interacting protein to the protein of interest
In meiosis I sister chromatids aren’t separated, what does happen during this stage
Homologous chromosomes pair up and crossing over takes place. These cells will be haploid with each homologue represented by two sister chromatids
Explain how deamination of cytosine is achieved
The NH2 group attached to the purine ring of cytosine at position 4 is replaced with a carbonyl group
What is the significance of tandem zinc finger repeats usually found in proteins
Tandem zinc finger repeat domains occur as part of larger DNA binding regions. Proteins with more zinc fingers can recognise longer sequences
Which groove of the DNA do protein interact with and why
DNA binding proteins interact with the major groove of the DNA because the minor groove is too narrow
What is significant about non-coding RNAs
Non-coding RNAs serve structural and enzymatic functions, acting more like proteins
What is indicated by linkage analysis experiments that reveal few offspring that show both the mutation and the marker
It means that the mutation and the marker lie close to each other and hence crossing over is rare
Cells often express different genes in a diseased state, T or F
T
Homologous recombination is required to mediate genetic recombination and diversity created in meiosis, T or F
T
Describe the chemical mechanism for the addition of a new nucleotide to the growing polynucleotide chain
The 3’ deoxyribose sugar undergoes nucleophilic attack on the first phosphate bond of an incoming nucleotide trisphosphate
The supernatant produced in the final stage of differential centrifugation contains only viruses, ribosomes and large macromolecules, T or F
F – the final supernatant contains only pure cytosol
What is the benefit of isolating misfolded proteins
Stops them from interacting with other proteins in the cell
What is meant by PolyA
The RNA transcript produced by the non-retroviral retrotransposon is poly-adenylated
Histone acetyltransferases can modify many different lysine residues, T or F
T
Due to internal folding within the molecule and complimentary base pairing, an RNA molecule appears as a 10th of the length of the corresponding DNA sequence, T or F
T
What is meant by the primary sequence of RNA
The polyribonucleotide sequence
What are the regulatory domains of src-tyrosine kinase and to what do they bind to
SH2 binds to phosphorylated tyrosine residues and SH3 binds to other proteins involved in regulation
What is the advantage of creating genomic libraries
It contains the entire genome sequence including all genes and regulatory sequences allowing for the study of transcriptional regulation
Acetylation and methylation of core histone tails can occur simultaneously, T or F
F – methylation and acetylation are mutually exclusive and are competing modifications
Explain how mass spectrometry can be used to determine the identity of unknown proteins
The isolated protein is incubated with proteases to digest it into peptides. These small peptides are then allowed to run in a mass spectrometer which ionises them. The mass spec machine then compares the recordings from the peptide fragments with a database of all known peptide combinations in order to determine the identity.
The RNA molecule produced in transcription will have the same sequence as the DNA sequence that codes for that gene, T or F
F – it will have the opposite sequence to the template strand
Why is dideoxy terminator sequence no longer used
Requires a lot of time and effort to read off the gel base by base. It also requires the primers to be radioactively labelled and left over night on an X-ray film to allow visualisation of the strands
3D radioactive peptide fingerprinting involves exposing peptides to radioactive phosphate or ATP and then X-ray radiation to determine regions of the peptide that are phosphorylatable, T or F
F – this is 2D radioactive peptide fingerprinting
What can be said about the genes that control the cell cycle
They are extremely conserved and almost identical across the species
Explain how a Double Holliday Junction is created during genetic recombination in meiosis
Two chromatids exist with different alleles for two genes (AB and ab). Spo11 endonuclease makes an initial cleavage in chromatid to create a dsDNA break. Mre11 then carries out a 5’ resection to create a targeted dsDNA break with a 3’ overhang in the DNA molecule. RecA then mediates strand invasion by this strand on the other chromosome creating a heterocomplex/triple helix structure. DNA polymerase then synthesises across the gap between the end of the 3’ overhang from the invading strand and the 3’ end of the other chromosome. The 3’ end is then relegated with the original strand to create a double holliday junction whereby the invading strand from one helix is forced into interactions with the complimentary strand in the helix of the other chromatid and vice versa.
What can be said about the error rate in transcription
Its much higher than DNA replication
What is the role of SH3 domains
SH3 domains bind to proline-rich motifs (poly-proline regions) and acts as an adaptor domain to link proteins together
What is meant by a molten globule
A molten globule is the structure formed from the initial folding of the protein that achieves a roughly correct conformation
Which regions of pre-RNA are spliced out but only in eukaryotes
Introns
Motor protein transport of organelles doesn’t require ATP, T or F
F – it does
What accounts for the high level of stability in leucine zipper domains
The hydrophobic leucine residues that extend into the space shared between intertwined helices are tightly packed together
What is the role of elongation factors in translation fidelity checking
Elongations promote translation and improve its accuracy
What is particularly useful about the vectors used in short sequence DNA cloning
Plasmids have their own very active origin of replication which usually results in 50 copies of the plasmid being make in each bacterium
DNA transposons make up the largest proportion of transposable DNA elements in the human genome, T or F
F – they only account for 5%
Which RNA polymerase transcribes all tRNAs, 5S rRNAs and other snRNAs
RNA Polymerase III
How can gene prediction software be used to identify genes in a sequenced region of DNA
Prediction software can be used to analyse newly sequenced regions of DNA scanning for promoters, start/stop sequences and intron splice sites which would implicate a gene.
How do retroviral retrotransposons act
Function via the production of an RNA intermediate
What are the two ways of predicting the secondary structure of a protein
Can be determined from the primary structure de novo or by alignment with other proteins of known structures using a website database
Describe the structure of the 10nm chromatin fibre
Single strand of DNA coiled around core nucleosomes
How does the telomerase enzyme act to create and maintain these telomeric repeats at the end of linear chromosomes
Telomerase is a ribonucleoprotein with an intrinsic RNA component containing the complimentary RNA sequence to the telomeric repeat sequence (AAUCCC). This RNA component acts as a template on which telomere repeat sequences are synthesised in a step-wise process known as the telomere shuffle. This allows addition of multiple TTAGGG repeats to the 3’-OH at each telomere.
How many amino acids are there
20
The hydrogen bonds that form in ? sheets form between polypeptide chain and are referred to as intra-chain hydrogen bonds, T or F
F – the hydrogen bonds do form between polypeptide chains but are referred to as inter-chain hydrogen bonds
Give an example of a protein that is post-translationally modified by hydroxylation and which amino acid is hydroxylated
Proline residues in collagen are hydroxylated
Explain the process of equilibrium sedimentation type density centrifugation
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. This causes the organelles to deposit in the sucrose depending on their density with heavier, denser organelles depositing at the bottom of the tube and so on etc.
Retrotransposons are abundant in invertebrate genomes, T or F
F – they are abundant in vertebrates
What is the universal start codon and what amino acid does it code for
Start codon AUG – Methionine/Met/M
How were restriction enzymes first identified
Restriction enzymes are part of a naturally occurring defence mechanism that digests foreign bacteria
Eukaryotic DNA replication is monophasic, T or F
F – its biphasic
How is wobble base pairing achieved
Modification of bases within the anticodon. Deamination of guanine creates inosine which can pair with uracil, cytosine or adenine
What two methods are there of repairing dsDNA breaks
Non-homologous end joining, homologous recombination
Describe the propagation of translation after the ribosome has fully assembled
Once this AUG has been reach and eIF’s have dissociated another aminoacyl tRNA bound to Elongation Factor-Tu binds to the vacant A site of the ribosome. If the anticodon of this aminoacyl tRNA doesn’t match the mRNA codon then this tRNA is ejected/falls off. Once the tRNA with the correct anticodon binds to the A site, EF-Tu hydrolyses its bound GTP and dissociates. The ribosome then catalyses formation of a peptide bond between the two amino acids. Following this the ribosome undergoes a conformational change that shifts the initiator tRNA into the E site of the ribosome. The now vacant P site is filled by the newly bound tRNA and EF-G binds to the ribosome. GTP hydrolysis by EF-G switches the ribosome back to being able to accept the next incoming rRNA. This process repeats until a stop codon is reached.
What is G0 phase
A quiescent, non-dividing phase
How are forward genetic screen carried out
An organism is taken and its genome is randomly mutated using a mutagen. Then, interesting phenotypes are looked for in the offspring of the mutagenized animals. Once the interesting phenotypes have been identified the next stage is to identify the actual gene that the mutation is in
What is coupled mass spectrometry
Coupled mass spectrometry involves subjecting the peptide fragments to further digestion and then fragmentation using an electric field. The mass spectrometer can than analyse and determine the amino acid composition of the peptides.
What happens during meiosis II
Chromosomes line up on the metaphase plate and sister chromatids are separated
The ligation process is rendered energetically highly favourable by the conversion of pyrophosphate (PPi) to 2Pi by pyrophosphatase, T or F
T
How can a helical structure be implied by the amino acid composition at certain positions
Hydrophobic (leucine or valine) residues at every 4th or 5th position in the primary sequence implies a helix
How specifically does elongation factor-1, EF-1 improve the accuracy of translation
After the anticodon has bound it causes two delays before the peptidyl transferase can act. Firstly it ensures that it must have hydrolysed its bound GTP and then it must have dissociated from the tRNA. These lags allow time for incorrect tRNAs to fall off.
Which metal ion(s) bind to catalytic metal ion domains
Fe2+ or Cu2+
What is meant by reverse genetics
Uses a known mutated gene sequence to observe effect on phenotype.
Explain the process of Western Blotting
The separated proteins from SDS-PAGE are transferred to a membrane using electrical current that moves the negatively charged proteins to the positively charged membrane. Then an excess of primary antibody is added to the membrane which specifically binds to the target protein. After unbound antibody is washed off an excess of a secondary antibody is then applied to the membrane. This secondary antibody binds selectively to the Fc region of the primary antibody. It also contains an enzyme bound it which when activated leads to the production of a fluorescent or coloured product.
In general transcription activators promote RNA polymerase II recruitment, T or F
T
Describe how transmission electron microscopy can be used to infer protein structure
The protein of interest is placed on an EM grid and spiked with a solution containing a heavy metal shading that is impermeable to electrons. Electrons are then fired at the protein and a negative stain is produced. Image analysis is then employed to build-up an average structure
What is the main advantage of NMR
The process is very dynamic and can be used to determine protein structure in solution
Describe the composition of telomeric repeat sequences
Telomeres consist of a hexanucleotide repeat sequence (TTAGGG)
Explain how the action of eIF-4G and eIF-4E act as a checkpoint in translation
eIF-4E and eIF-4G only bind to mRNA that is capped and has a polyA tail. This acts as a checkpoint for broken mRNA
Cyclins are expressed at constant levels during the cell cycle, T or F
F – cyclins are transiently expressed and are synthesised and degraded throughout the cell cycle
Explain the link between protein misfolding and aggregation
Misfolding of proteins often leads to exposure of hydrophobic regions which is what causes aggregation
How many different codons are there for serine, and how many different tRNAs
6 different codons but only 3 different tRNAs
Cyclins are a class of protein involved in the regulation of the cell cycle, explain how they interacts with their targets
Cyclins bind to cyclin-dependant kinases (Cdks) to activate them
What accounts for the end replication problem during DNA replication
The need for an RNA primer for initiation of DNA synthesis. Ribonuclease H removes the last RNA primer in the linear chromosome sequence however DNA polymerase can no longer extend the DNA sequence. This would result in a gradual reduction in the length of replicated DNA by the length of one primer with each subsequent replication
Describe the structure of a primer and how they are made
A primer (oligomer) are short, roughly 20 nucleotide single stranded DNA sequences that are usually synthesised chemically. To synthesise primers, a small part of the DNA sequence of interest must be known. This is usually part of the vector sequence in which the target sequence is immediately integrated into after.
Which type of transcription factors will bind to methylated lysine 4 and arginine 17 residues
Transcriptional activators containing PHD fingers
How can splicing explain how the same gene can produce different proteins when translated
The mRNA transcript can be spliced in different ways which accounts for differences in the proteins produced
Explain the molecular mechanism of X-chromosome inactivation
X-chromosome inactivation involves the synthesis of a non-coding RNA known as Xist from the X-inactivation centre (XIC) on the chromosome destined for inactivation. Xist RNA binds to the X chromosome and acts as a recruitment signal to promote the formation of silent chromatin. This is achieved by recruitment of histone modifying enzymes and other Polycomb Group components and leads to the Histone-3 lysine 27 and H3K9 methylation of core Histones in X chromosome chromatin
What reaction supplies the energy required for DNA synthesis
Breakage of 2 high energy phosphate bonds
Define the primary protein structure
Individual linear sequence of amino acids
How do SSBs ease replication fork progression
They act to prevent hydrogen bond formation between complimentary base pairs within the same ssDNA strand by binding to the sugar-phosphate backbone and allowing easy progress of polymerase
What happens in patients with mutations in genes needed for homologous recombination
Homologous recombination is defective and thus these cells become critically dependant on other pathways for correct DNA repair i.e. base excision and nucleotide excision repair
What is the results of the formation of pyrimidine dimers
This arrests DNA replication or can cause mis-reading of the DNA sequence by DNA polymerase
What is the main difference between meiosis and mitosis
Meiosis resembles mitosis except there are extra steps that segregate homologous chromosomes
Sex chromosomes don’t cross over, T or F
F – they behave like homologues during sperm formation due to small regions of homology
CD also give information on the arrangement of proteins, T or F
F – it cannot give arrangement information
Near to which genes have human DNA replication initiation sequences been found
LMNB2 (laminin B), MYC and HBB (Haemoglobin B)
To sequence large fragments of DNA greater than 1kbp, plasmids are used as a vector, T or F
F – bacterial artificial chromosomes are used
What is the role of EZH2 in development
EZH2 is a gene required to repress Hox gene expression in a specific anterior-posterior fashion
Which enzyme is responsible for breaking the hydrogen bonds between complimentary base pairs
DNA helicase
Which DNA damage(s) is base excision repair used for
Deamination and depurination
How can CD be used to look at changes in protein structure
CD can be used to investigate the effects on protein structure due to changes in pH and temperature. For example, increasing pH increases helix formation whilst decreasing ? sheet formation. In addition, it can be used to visualise protein unfolding due to increasing temperatures.
During DNA duplication, each chromosome pair (maternal and paternal) is duplicated to give rise to sister chromatids, T or F
T
DNA cloning involves ligation of DNA fragments into vectors. What vectors are commonly used
Plasmid vectors – small circular, extra-chromosomal DNA that occur naturally in bacteria
What is the problem with gene prediction software
You cannot be sure that predictions are always true and a gene has or hasn’t been identified
Structurally and in terms of proteins encoded by them, how are retrotransposons similar to retroviruses and what is a significant difference
Retrotransposons contain DNA sequences that code for envelope and capsid proteins just like retroviruses do. However, these proteins are usually defective but without preventing the replication capability of the retrotransposon
Palmitoylation and farnesylation are examples of lipidation, T or F
T
What is meant by a hypermorphic mutation
Mutation that confers a gain of function of the gene product. This is caused by the overexpression of a transcription unit or overactivity of a gene production.
When was the human genome started and completed
Started in 1990, finished in 2003
Explain the process of differential centrifugation
Differential centrifugation starts with a cell homogenate that is centrifuged at low speed. This creates a pellet contains the heaviest cytosolic components/organelle such as nuclei, whole cells and the cytoskeleton. The supernatant from this centrifugation is then separated and centrifuged again at medium speed and second pellet is produced this time containing mitochondria, lysosomes and peroxisomes. The second supernatant is the centrifuged again at high speed to produce a pellet containing microsomes and vesicles. This supernatant is then centrifuged in the next step at very high speed to produce a pellet containing viruses, ribosomes and large macromolecules. In the final step the supernatant from this stage is spun again at a very high speed for a very long time to produce a supernatant containing only pure cytosol
How many sub-domains are contained in the src-tyrosine kinase
4
The position of an amino acid in the primary sequence enables you to predict the secondary structure of a protein, T or F
T
How does cyro-electron microscopy enable the derivation of average protein shape
Vitreous ice is used to preserve/freeze the protein specimen. Electrons are then shone onto the frozen specimen and the average shape of the protein is determined.
How many generations does it take to make a particular mutation homozygous
3 generations; Po, F1 and F2
What is meant by a cDNA library and what is its advantages
cDNA library is created from the mRNA transcribed within a specific cell or tissue. This allows for the study of disease to identify which genes are expressed in a diseases tissue compared to a normal healthy tissue
How can electron microscopy be used to directly visualise interacting proteins
EM uses a negative stain or vitreous ice (cyro-EM) to preserve the specimen. Image analysis is then employed to build up an average protein structure. EM allows visualisation of changes in the protein i.e. if it becomes thicker in certain parts when a binding partner is added. This allows you to determine where this potential binding partner is binding - which domain
What is meant by forward genetics
Forward genetics is the method by which you identify the function of a gene and then identify the gene itself
Which specialised sequence within chromosomes is the DNA sequence at which the kinetochore assembles to mediate chromosome segregation at mitosis and meiosis
Centromeres
Give an example of a permissive transcription factor
TATA binding protein (TBP)
Not all of the genetic information in eukaryotes is encoded in the nucleus, where else is some genetic information stored and how
Some genetic information is contained in the mitochondria and chloroplasts in the form of small circular chromosomes
How are the progeny of the cell that silences one of the X chromosomes in somatic female cells affected
The silencing decision is propagated clonally and all progeny of each cell in which the silencing decision was taken inherit the same silenced X chromosome
Give an example of a change in the non-coding sequence of a gene transcript
Mutations that lead to the deletion of an exon
Explain the process of DNA excision repair
The damage base is first removed by DNA glycosylase which cleaves off the inappropriate base(s). The deoxyribose sugar is then removed by an enzyme known as apurinic/apyrimidinic endonuclease. Lastly the phosphate is removed by the phosphodiesterase enzyme. This leads to a ssDNA break in the polynucleotide chain. This gap is then recognised by DNA polymerase which then extends over this gap which acts as a primer template junction. Finally, DNA ligase uses ATP hydrolysis to provide the energy required to seal the nick
What are the three types of transposons
DNA Transposons, Retroviral retrotransposons, Non-retroviral PolyA retrotransposons
What are the three main methods of DNA repair
Base excision repair, nucleotide excision repair, homologous recombination
What can make DNA molecules susceptible to dsDNA breaks
If the DNA has already suffered a ssDNA break due to base excision repair or nucleotide excision repair. This may weaken the DNA structure
How can CD be used to determine protein refolding capabilities
Cooling the protein down can be used to determine the ability of the protein to refold by seeing if it can regain its characteristic CD spectrum
What two features are required for synthesis of RNA primers
DNA template strand and nucleotide trisphosphates
How does Eukaryotic replicator selection occur
Origin replication complex (ORC) binds to the replicator sequence. Helicase loading proteins then bind to ORC to convert the single stranded replicator sequence into a pair of replication forks. Mcm2-7 then also binds to complete formation of the pre-RC
Explain how linkage analysis allows the identification of a particular gene
Linkage analysis involves analysing recombination between an identified allele and a known marker on the same chromosome. This enables the determination of whether the gene and marker are linked in any way. Carriers of the mutation are crossed with carriers of the marker and the number of offspring where crossing over has occurred in, are counted. These are the number of offspring that exhibit both the mutation and the marker. The greater the distance between the gene and the marker on the chromosome the more frequent that crossing over events will occur and the greater the number of offspring produced with both attributes.
Where in the cell cycle does replicator selection and formation of the pre-replicative complex occur
G1
How can you calculate the equilibrium constant for the interaction between two proteins using radioactive ligand binding data
K = [AB] / [A][B] = kon / koff
DNA transposons are also powerful mutagens, T or F
T
What percentage of the F2 generation of flies produced by a forward genetic screen will show a phenotype if there is one
A quarter
Which end of the mRNA strand is polyadenylated
3’ end
What features of the cohesive/sticky ends allow ligation
The ability of them to hybridise based on complimentary base pairing
Recall the general and word equation for the reaction coupled to DNA ligase activity that accounts for its irreversible nature
PPi –> 2Pi + Free energy. Pyrophosphate is converted to two molecules of inorganic phosphate by pyrophosphatase, which also releases energy
What is the main benefit of carrying mobile genetic sequence information around
Acts as a genome shuffling mechanism that breaks up and reassembles the genome, providing new combinations of DNA sequences and facilitating rapid genomic evolution
What is meant by the term transposon
Mobile genetic elements that can replicate themselves and jump around the genome
What is meant by blunt restriction enzymes
Restriction enzymes that cut the DNA flush
Once the construct has been designed how is this used to create offspring
The construct is then introduced into mouse embryonic stem (ES) cells using cell culture techniques. The cells DNA repair machinery then recombines the construct into the mouse ES cell genome
Which amino acid residues end up in the centre of the proteins secondary structure and why is this
Non-polar side chain amino acids end up in the centre of the proteins structure forming hydrophobic core regions
What is meant by the term permissive transcription factors
Permissive transcription factors are general transcription factors necessary for all transcription and are non-regulatory. These bind at the promoter sequence of the gene and are ubiquitously expressed. Binding of permissive transcription factors to the promoter helps the polymerase machinery to find the start site
Progressive sequencing is one method of sequences genome sequences greater than 1kbps, explain how this process works
You start with a genomic library and genomic sequences. Automated sequencing is then used to determine the 1000bps at either end of the genomic insert, adjacent to the vector sequence. The ends of each clone are sequences using primers that have been designed for the vector sequence. Once the first 1000bps at either end of the genomic insert have been determined, primers are then designed for each of these sequenced regions. Another round of sequencing is then performed to determine the next 1000bp sequence of the genomic insert adjacent to the already determined regions. This process repeats until the sequences produced overlap in the middle.
What is significant about the enzyme required by DNA transposons to move throughout the chromosome/genome
Transposase is encoded by the DNA transposon itself
How does indirect ELISA differ from the direct approach
Indirect ELISA uses a secondary antibody that binds to the primary antibody to visualise interaction with the target protein. This secondary antibody is the one that contains the tag/label
What mechanism do DNA transposons move by
Cut-and-paste – without self-duplication
Explain the major differences between the two types of topoisomerases
Type I Topoisomerases nick and release one of the 2DNA strands and effective remove one turn in the molecule. This is an ATP-independent reaction. Type II Topoisomerases nick a reseal both DNA strands by causing dsDNA breaks effectively removing two turns of the supercoiled helix. This reaction is ATP-dependent
What is meant by complementation testing
Complementation testing is used to determine if different mutations that have the same phenotype are alleles of the same gene or if these mutations in fact lie in separate genes.
Homologous recombination also isn’t a perfect repair mechanism, T or F
F – homologous recombination provides a perfect repair of dsDNA breaks and is an accurate and preferred method of repair
What is the purpose of the polyA tail
The polyA tail serves as a signal to stop translation
What is the purpose of boiling protein samples with mercaptoethanol in the first stage of SDS-PAGE
Boiling with mercaptoethanol breaks the disulphide bonds between cysteine residues
DNA polymerase can only synthesis in a 3’ –> 5’ direction, T or F
F – vice versa
The amino acid sequence of proteins is thought to have evolved over time to promote formation of the molten globule, T or F
T
Explain how CD works
CD spectroscopy uses far-UV radiation (190-250nm) to reveal the secondary structures of proteins. Each type of secondary structure has a unique and characteristic CD spectrum. ? helices have two-peak spectrums at 208 and 225nm whereas, ? sheets give a single peak between 216-218nm. Random coils also have a unique CD profile
What chemical variables explain the irreversible nature of DNA synthesis
Equilibrium constant, Keq of the magnitude of 105 and the Gibb’s free energy, ?G = -7kcal mol-1
Describe the structure of the 30nm chromatin fibre
Causes by a supercoiling of the 10nm chromatin fibre into a superhelix
Sister chromatids are then segregated between the two daughter cells, T or F
T
Explain how NMR can be used to determine protein structure
Specific isotopes of carbon and nitrogen (13C and 15N) are introduced into the protein of interest by growing bacteria in a medium containing nutrients with these specific isotopes only. The subatomic particles of these isotopes possess a quantum-mechanical spin. These spin vectors are aligned with a large magnetic field in a number of 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.
What are the advantages of using yeast to study the cell cycle
Rapid division rate <1hr, cell cycle control genes almost identical to human, can be grown as haploids or diploids
What is meant by DNA replication being semi-conservative
Each new daughter strand produced in DNA replication consists of one parental helix and one newly synthesised DNA strand
Recall how Zn2+ ions interact with zinc finger domains
Zn2+ ions are coordinated tetrahedrally with the zinc finger domains. This is achieved by interactions with 2 cysteine residues from the ? sheet and 2 histidine residues from the ? helix
What prevents the actin filament from sliding back after being released by the myosin motor head
There are many other myosin molecules also attached to it that hold it under tension
The large ribosomal complex contains the peptidyl transferase enzyme, T or F
T
Often after separation based on isoelectric points, proteins are then separated using electrophoresis to distinguish proteins based on size, T or F
T
Protein databases provide a wealth of information and predictions based on sequence alignment, domain composition, post-translational modification, protein-protein interactions and structures, T or F
T
Describe the structure/composition and role of centromeres
Higher order repeat sequences that contain subsets of repeat sequences called alpha-satellites. Centromeres facilitate chromosome segregation during cell division
Explain the structure of muscle myosin
Muscle myosin is a dimer with two identical motor heads which act independently. Each myosin head has a catalytic core and an attached lever arm. A coiled-coil rod ties the two heads together, and tethers the myosin heads to the thick myosin filament
What is the name of the restriction endonuclease that recognises the GAATTC sequence
EcoRI
Describe the quaternary structure of the leucine zipper domain
Leucine zippers contain two intertwined ? helices
How many genes are there in the human genome
23000
The tertiary structure of RNA refers to its interactions with other RNAs, T or F
F – tertiary structure is the RNA strands 3D conformation
What are the downstream implications of changes in the non-coding sequence of the transcript of a gene
These may effect RNA splicing, stability or folding
Describe how Edman degradation can be used to obtain the primary protein structure
Using phenyl isothiocyanate (PCT) to bind to and break away the terminal amino acid in a step by step process you can determine the unique sequence by high performance liquid chromatography (HPLC). This is done based on the molecular weight of each residue
What can be said about the direction of hydrogen bonds in ? helices
They are vertical hydrogen bonds
What are the three functions of snRNAs
Recognise 5’ donor and branch sites, bring sites together and catalyse cleavage
Peroxisomes, lysosomes and mitochondria are contained in the second pellet during differential centrifugation, T or F
T
For fragments between 300kbps and 3Mbps, what vector if best suited
Yeast artificial chromosome
eIF-4G, eIF-4E, small ribosomal complex binds to the polyA tail of the mRNA strand, T or F
F – the complex binds the capped head of the mRNA
In RNA A pairs with U and C pairs with G however it is possible for unusual base pairing such as G with U, how is this described
Non-Watson-Crick pairing
What is significant about the tails of core histones
Nucleosome core histones have N-terminal lysine rich tails which project radially from the core. These can be reversibly covalently modified
What do SH3 domains bind to
Proline-rich motifs
Which residues are commonly acetylated by histone acetyltransferases
Lysine residues
What is the role of topoisomerases
Prevent the DNA from becoming tangled and supercoiled during DNA replication
Which residue(s) are often acetylated by acetyltransferase enzymes
Lysine
What is the result of changes in the regulatory sequences of the DNA
These affect transcription and occur in regulatory sequences such as enhancers
CD spectroscopy measures the differential absorption of linearly polarised light, T or F
F – measures the differential absorption of circularly polarised light
DNA binding proteins form stable interactions with the DNA sequence to which they bind, T or F
F – the interactions between DNA and DNA binding proteins are not stable
New amino acids are added to the N-terminus of growing polypeptide chains, T or F
F – they are added to the C-terminus
Which two amino acid residues have low helix forming propensities
Proline and glycine
Describe the process of gel filtration chromatography
Cytosol that has previously been centrifuged is loaded into a column containing a solid matrix of beads of a certain size. A neutral buffer and solvent is then added to the solution which acts to push the cytosol down the column. Larger proteins have a smaller volume to move through, due to their size, than smaller proteins. Hence larger proteins will leave the column first due to the sieving effect or the pore size between the beads. The different fractions of the cytosol can thus be collected drop-by-drop over time to separate the mixture.
PolyA tails can be up to 200 residues in length, T or F
T
Explain the enzymes involved in the regulation of Cdk activity
Wee1 kinases is responsible for the phosphorylation and inactivation of Cdk. Whereas Cdc25 phosphatase removes a phosphate group from the inactive Cdk hence activating it. The interplay between Wee1 kinase and Cdc25 phosphatase determines that activity of Cdks.
What features define transposons and are the site for enzyme binding
Short simple sequence repeats that lie at each end of the transposon where transposase binds
How are modified ES cells that contain only the knockout and NEO genes isolated from those that contain also contain the TK gene and those cells that only contain the functional target gene
The cells are cultured in a medium containing neomycin and GANC. The cells that haven’t incorporated the construct will be unable to survive in the antibiotic medium as they don’t contain the resistance gene. Similarly, those cells that have also incorporated the TK gene will be unable to survive also despite having the NEO antibiotic resistance gene. This is because the TK gene makes the GANC medium toxic to them. This will leave only those ES cells containing the construct with the knockout target gene and the NEO gene.
Phage display is referred to as a genetic technique to investigate protein interactions, explain how it works
Random peptide sequences from a peptide library are integrated into a phage plasmid between restriction sites within the coat protein region. This means that the potential interacting peptides will be expressed in the coat of the virus allowing for potential interactions with the protein of interest. A solution containing the millions of potentially interacting peptides are added to the target protein. Unbound phages without interacting peptide can be removed using a buffer wash whilst interacting proteins will bind to the protein of interest. Addition of high concentration salt solution can the allow elution of bound phages that indicate interacting peptides. The phages that bind to the protein of interest can then be isolated, multiplied, DNA extracted and the sequence of the interacting peptides determined.
What physical phenomenon of atoms/molecules drives the random movement of kinesin heads
Brownian motion
How has the dideoxy terminator sequencing method been improved
Rather than labelling the primers the ddNTPs are labelled themselves, each base with a different colour
Recall the equation that calculates the genetic distance between mutation and marker in linkage analyses
cM = R/T x 100 (genetic distance = number of recombinant gametes/total number of gametes)
Describe the structure of a triple-coiled-coil protein
Another stable structure formed by ? helices. This is where 3 amphipathic helices twist around a central axis with the hydrophobic side chains of all three exposed in the centre of the structure. This creates a stable hydrophobic core
Which base is primarily affected by UV light and more prone to form dimers
Thymine
Release of eIF-2 initiates translation, T or F
T
What are pyrimidine dimers and what causes them
Pyrimidine dimers are caused by the covalent linkage of benzene rings in two adjacent pyrimidine bases. This is usually caused by UV light
Explain the process of nucleotide excision repair
An excision endonuclease enzyme cleaves the single strand of the DNA containing the defect by cleaving either side of the dimer etc. This creates a polynucleotide fragment from the DNA molecule that contains the defect which is then removed by DNA helicase. DNA polymerase then extends the primer template junction created to replace the excised sequence. This is followed by resealing of the nick mediated by DNA ligase.
Which two amino acids can be phosphorylated
Serine and threonine
What are the results of mutations in the coding sequence of a gene
May alter amino acid composition which can impact folding
What do chromosomes consist of
Hexanucleotide repeat sequences that create a 3’ overhang in the linear chromosome
Explain how different resolutions of the Double Holliday Junction account for whether homologous recombination occurs or not
If the internal strands are broken and re-joined than the same DNA strands are broken and re-joined at each junction and recombination is not achieved as each chromatid will contain the same sequence of alleles it did prior to cleavage. If external and internal strands are broken and re-joined then different DNA strands are broken and re-joined at each junction. Hence recombination is achieved. This recombination of allelic forms results in chromatids with different combinations of alleles (Ab and aB)
What two effects can ubiquitination have and how are these different pathways encoded
Poly-ubiquitination marks proteins for degradation whereas mono-ubiquitination directs protein recycling
In what plane do the hydrogen bonds between ? sheet polypeptide chains occur
Horizontal hydrogen bonds
What localised structure is formed by the breaking of hydrogen bonds between complimentary base pairs in the parent strand
Replication fork
What is meant by the abbreviation ORF
Open reading frame
What are EF Hand domains
Ca2+ binding domains that have a regulatory or structural function
Uvr genes are homologues of the XP genes found in yeast and are also transcription coupled, T or F
T
What is the most frequency types of DNA damage
Hydrolytic depurination and deamination of bases
The vectors used in DNA cloning usually contains antibiotic resistance genes in bacteria and can be transferred onto the progeny and between adult bacterial cells, T or F
T
Explain how affinity chromatography is used to investigate interacting proteins and how it is achieved using pulldown proteins
Recombinant technology is used to create a fusion protein consisting of the protein of interest and glutathione-S-transferase (known as a pulldown protein). The fusion protein and cytosol is then added to a column containing glutathione coated beads. The GST fusion protein will bind to the glutathione coated beads together with any proteins in the cytosol that interact with the protein of interest. Once the cytosol has passed through the column and interacting proteins have bound together with the protein of interest to glutathione beads, a solution of free glutathione is added to the column to elute all of the bound proteins. These can then be separated using SDS-PAGE and identified using mass spectrometry.
Explain the enzyme-linked immunosorbent assay technique to determine protein interactions
The antigen, i.e. protein of interest is attached to a 96 well plate. Then a primary antibody is then added to the plate that binds to the protein of interest. In direct ELISA, this primary antibody carries a label that allows it to be visualised.
What is the main limitation of automated sequencing
Automated sequencing is restricted to 1000-1500 base pairs at a time
Leucine zipper domains are protein-protein binding or protein-DNA binding domains, T or F
T
There are over 50 possible modifications of the bases in tRNAs, what is meant by psi and D bases
psi corresponds to pseudouridine and D is dihydrouridine
Explain the role of RNA primers in the synthesis of DNA
DNA polymerase requires an RNA primer in order to synthesise a new DNA strand. RNA primers supply DNA polymerase with base-paired chain ends to add new nucleotides to.
What are the three possible stop codons that signal the end of the ORF
UGA, UAG and UAA
Explain RNA polymerases role in synthesising the polyA tail
RNA polymerase contains a tail that is highly phosphorylated. The negative charge of these phosphates resembles the RNA backbone negative charge. This serves as a docking point for RNA binding proteins which bind to particular RNA sequences known as polyA signals (AAUAAA). Cleavage stimulating factor (Cstf) and cleavage/polyadenylation specific facto (CPSF) jumps off polymerase II when this sequence is detected and bind to the polyA signal and cleave the RNA. This allows PolyA polymerase to synthesise a polyA tail
What is significant about sliding clamp proteins across Eukaryotes
Extremely highly conserved
Telophase corresponds to cytokinesis, T or F
T
How does introduction of the construct into mouse ES cells lead to the production of 3 different cell populations
One population of transformed ES cells will have undergone homologous recombination and contain the knocked-out target gene with the NEO gene in between the homologous arms. Another population that will have undergone non-homologous recombination will have these genes and the downstream TK gene also. The final population will contain ES cells that haven’t integrated the construct at all
What accounts for the similar structure seen in all tRNA molecules
Internal base pairing
What specialised DNA polymerase enzyme is responsible for replication of telomeres
Telomerase
What gives the leucine zipper its name
Many of the hydrophobic residues that extend into the space shared between intertwined helices are leucines
Chromatin is a protein, T or F
T
What ways can transcriptional repressors act to decrease gene transcription
Compete for activator binding sites, prevent bound activators from functioning, keep transcriptional activators and transcription machinery away from the start site and also reverse effects of transcriptional activators by creating dense transcriptionally inert chromatin. Finally, they can also recruit enzymes such as histone demethylases and deacetylases as well as methyltransferases
What aspect of the SH2 domain provides specificity
Specificity comes from the interaction between the phosphate of the phosphotyrosine and consists of mainly ionic interactions between the negatively charged PO42- and positively charged amino acids. Some hydrogen bonding also contributes
How are Cdks further regulated other than the action of cyclins
Cdks are also regulated by their phosphorylation state. Active Cdk contains 1 phosphorylated serine residue whereas inactive Cdk has 2 phosphorylated serine residues. Unusually phosphorylation is an inhibitory signal in the case of Cds.
mRNA accounts for the majority of RNA, T or F
F – RNA only accounts for between 3-5% of all RNA
How can the ability of yeast to be haploid or diploid be harnessed to study the cell cycle
If you want to study a gene involved in cell cycle, manipulation will have a very deleterious effect on that organism. If you have carriers of the deleterious gene in diploid state and then switch the yeast to haploid when you want. Thus, diploids can be used to maintain lethal mutations that are then studied as haploids
What structures are referred to as the building blocks of chromatin
Nucleosomes
What are the four different ways that the genome can be compared online
Chromosomes can be determined, genes can be predicted, regions of protein homology can be seen and expression data can be provided by comparing expressed sequence tags from a cDNA library
What are SNPs
Single polynucleotide polymorphisms are single base pair changes that are frequent in the population, varying from individual to individual but without conferring a disease phenotype
Once the desired ES cells have been obtained that contain the gene knockout, how are mosaic mice produced
The selected cell line containing the knockout gene are reintroduced into mice embryos from a different genetic background to the original ES cells. These embryos are then implanted into back into the female mouse and leads to the production of a first-generation mosaic mouse. These mice contain a mixture of cells from the transformed stem cell line and cells from the mother.
Which technique is useful for visualising chromosomes in interphase nuclei
Chromosome painting
How is the starting cell homogenate obtained in differential centrifugation
Blending, sonication or grinding with a pestle and mortar
How many base pairs are there approximately in the human genome
3.2x109 – 3bn
Two which terminus of the ? sheet do the arrows seen in ribbon diagrams point to
Carboxyl (C) – terminus
Which class of amino acid residues extend outwards from each helix into the space shared between them
Hydrophobic side chains
Levels of Cdk remain constant throughout the cell cycle, T or F
T
Recall the structure of a mature mRNA from start to finish
Methyl guanosine cap –> 5’ UTR –> START –> Coding Sequence –> 3’ UTR –> PolyA Tail
How can the yeast-2-hybrid screening technique be used to identify interacting proteins
Mix the prey and bait proteins produced by the yeast. The bait will bind to the reporter gene (HRP, GFP, ?Gal etc.) promoter via the fused binding domain. Prey fusion proteins containing protein(s) that interact with the protein of interest will bind to the bait via the protein of interest. Presence of both the BD and AD will lead to regulation of the reporter gene. The regulation of the reporter gene can then be used to identify the which yeast cells have the correct combination of interacting proteins. You can then isolate the plasmid from the yeast and sequence the plasmid to identify the gene the codes for the interacting protein
If a protein starts with methionine, you can determine that that is the start codon, T or F
T
How do restriction enzymes generally cut the DNA
Generally they cut the DNA leading overhangs known as sticky ends
Why is it often seen the 50kDa proteins are found in the middle of an SDS-PAGE
50kDa corresponds to the average proteins molecular weight (due to 500 residues) and so with most proteins being found in and around this weight it is a reasonable middle of the SDS-PAGE
Describe the composition of the ribosome in which protein synthesis/translation occurs in
The ribosome is composed of two different subunits. The complex consists of about 50 ribosomal proteins and several ribosomal RNAs (rRNAs)
Specific/regulatory transcription factors don’t have to bind directly to the gene which they regulate, T or F
T – they can exert their regulation of transcription by binding to a regulatory complex
What is the restriction or start point
A point in the cell cycle after G1 phase that determines the commitment of the cell to S phase and the completion of the rest of the cycle to G1 again
Methylation of core histones creates binding sites for transcriptional activators that contain what kind of domain
PHD finger domains
What is unique about the bases contained within tRNAs
The bases are highly modified to allows more specific interactions with the protein counterparts
How do myosin and actin filaments interact
Myosin heads initially contain bound ADP and phosphate, and have weak affinity for actin. Once one of the heads docks properly onto an actin subunit, phosphate is released. Phosphate release strengthens the binding of the myosin head to actin, and also triggers the force-generating power stroke that moves the actin filament. ADP then dissociates, and ATP binds to the empty nucleotide binding site. This causes the myosin head to detach from the actin filament and ATP hydrolysis occurs. This re-cocks the lever arm back to its pre-stroke state. The coiled-coil arm thus stores the energy released by ATP hydrolysis, and the cycle can repeat.
Explain how SDS-PAGE separates the different proteins
Once loaded into wells, the negatively charged proteins move towards to positive electrode. Smaller proteins will move faster/further in the gel than larger proteins and hence will progress more along the gel and will be closer to the anode.
What are the advantages using Xenopus laevis as a biochemical model when looking at the cell cycle
Easy to collects its eggs, rapid division rate, large sized eggs makes protein purification easier and they can be manipulated by injection of RNA or chemicals into the oocytes
Which RNA polymerase transcribes all protein coding genes
RNA Polymerase II
What can be used to mostly infer primary structure
The DNA sequence
What is are the main advantages of shotgun sequencing
Shotgun sequencing requires no thought and only requires a batch of primers predesigned for the vector sequence rather than needing the design and synthesis of multiple sets of primers. The process can thus be automated
Correct tRNAs once bound to the complimentary mRNA codon, don’t fall off, T or F
F – correct tRNAs do also fall off but at a much slower rate
Describe how 2D gel electrophoresis can be used to separate proteins
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.
What is the role of SDS in the separation of proteins in SDS-PAGE
Sodium Diethyl Sulphate is a negatively charged molecule that repels proteins and causes them to straighten out and thus allows the proteins to move easily through the polyacrylamide gel
What enzyme is required by DNA transposons in order to move throughout the genome in the way that they do
Transposase
Give an example of a method of displacing bound proteins in affinity chromatography
High salt concentrations
Why are dsDNA breaks especially dangerous
Large fragments of chromosomes can be lost
What are the names of the enzymes that reverse histone acetylation and methylation respectively
Histone deacetylase and histone demethylase
Reversibility of the acetylation and methylation of histones accounts for what attribute of epigenetic changes
Means that they can be removed – aren’t permanent
Most genetic screens are done for recessive alleles but domain forward screens can also be done, T or F
T
How can BLAST software be used to determine gene presence in sequenced DNA
Once the base sequence has been obtained you can use a computer to translate this in all 6 reading frames to derive the corresponding amino acid sequence. BLAST can then be used to search for similarities in the sequence that correspond to similar known proteins
What is the other mechanism of molecular chaperone action other than direct binding
Hsp60 class molecular chaperones put misfolded proteins into isolation. The hydrophobic entrance of hsp 60 binds to the protein and partially unfolds it. Then the GroES cap seals the protein inside for about 15 seconds to allow refolding
Will genes that are more transcriptionally active show higher or lower levels of acetylation
Higher
Explain the process of complementation testing
Complementation analysis allows the sorting of mutations into distinct groups that correspond to individual genes. Carriers of a mutation that has the same phenotype are crossed and the offspring produced from this cross are analysed. If ¼ of the offspring produced are homozygous mutants then the mutations fail to complement each other and they are mutant alleles of the same gene. If no offspring with the mutant phenotype are produced then mutations are said to complement each other and lie in different genes
What is the major disadvantage of X-ray crystallography
Extremely expensive (£200,000 in the lab, >£300m for professional synchrotron sources)
The binding constant can be determined from SPR using association and dissociate rate constants, T or F
T
Methylation can denote transcriptionally active or inactive genes depending on the loci of the residue. Determine whether methylation of lysine 4, 9, 27 and arginine 17 denote transcriptional activation or repression
Arginine 17 and Lysine 4 – transcriptionally active. Lysine 9 and 27 – transcriptionally inactive
dsDNA breaks are often caused by non-ionising radiation, T or F
F – they are caused by ionising radiation
What is meant by sandwich ELISA
The target protein antigen is captured by multiple antibodies. The primary antibody binds to the protein of interest but then another antibody that is also complimentary to the antigen, also binds to it. A secondary antibody is then introduced that binds to the primary antibody(s) and contains the tag/label
Explain how splicing occurs
The 2’ OH of an adenine branch site attacks the phosphodiester bond on guanine donor site. Cleavage at the donor site results in the formation of lariat. Next a 3’ OH in the guanine donor site attacks phosphodiester bond on a guanine acceptor site freeing the lariat which is then degraded
What is the name of the sequence in tRNA that binds to the mRNA codons
Anticodon loops
Other than determining the identity of unknown proteins, what other use is there for mass spectrometry
Analysing post-translation modifications such as serine/threonine phosphorylation. This causes a change in molecular weight which can be picked up by the mass spec
Which motor protein moves vesicles and other organelles towards the -end of the microtubules
Dynein
What are the roles of the subunit in the ribosome
The large ribosomal subunit catalyses polymerisation and peptide elongation whereas the small subunit facilitates the tRNA/mRNA interactions
Rox1 is a DNA binding protein found in yeast, how many different sites does it bind to
8 sites in three different yeast genes
How can you use temperature sensitive mutations in yeast to study the cell cycle
You can introduce mutations that only disrupt gene functions at particular temperatures into the yeast. Keeping the yeast at a low permissive temperature maintains gene function but switching to a restrictive (high) temperature enables you to study the genes function
Folding of a protein begins immediately after leaving the ribosome, T or F
T
Name two of the major classes of molecular chaperones
Hsp60 and hsp70
What is the name of the protein inhibits the activity of the cyclin-cdk complex
p27
What is the role of insulators and barriers in the control of gene expression
Insulators and barriers block regulatory sequences from affecting neighbouring genes and prevent enhancers from activating the wrong genes
Explain why non-retroviral PolyA retrotransposons are referred to as stripped down retrotransposons
They lack the envelope and capsid proteins encoded within the retrotransposon sequence
Lecture 1 Qn 18 Determine which representations of ? sheet protein structures the following images correspond to
Backbone, sticks, space filling, ribbon
What attribute of the ligation of DNA fragments makes it another example of an irreversible reaction
Like DNA synthesis ligation results in the formation of another molecule of pyrophosphate. This reaction is then coupled to a reaction the converts PPi to 2Pi
Explain how pairing of chromosomes is facilitated in meiotic prophase I
Pairing is facilitated by the synaptonemal complex as well as DNA base pairing between homologues
What is the name given to the groups of separate genes that when mutated produced the same phenotype
Complementation groups
What are the four different specialised sequences contained within chromosomes
Telomeres, centromeres, replication origins and kinetochores
Are hypermorphic mutations recessive or dominant
Dominant
Who discovered chromosomes in 1902
Sutton and Boveri
The irreversible nature of DNA synthesis accounts for the massive stability that is an inherent property of DNA molecules, T or F
T
What phenomenon is FRET said to rely on
Paired fluorescence
Once the colonies containing the transformed bacteria have grown on the antibiotic medium what is the next stage to produce an unlimited supply of the DNA sequence
Single colonies are lifted from the plate to start a liquid culture. The plasmids can then be easily purified from the bacteria and stored or analysed.
The helix-turn-helix domain is an example of a DNA binding domain, describe its structure and interaction with DNA
Helix-turn-helix domains consist of 2 ?-helices, one of which, the recognition helix interacts with the major groove of the DNA by making specific contacts with bases. Helix-turn-helix domains usually bind as dimers to palindromic recognition sequences contained within two consecutive major grooves
Explain how surface plasmon resonance can be used to investigate protein interactions
Light is shone onto a gold film with plasmon-induced evanescent electric filed which extends beyond the gold film by a distance that corresponds to the wavelength of the light shone onto it. Changes in the composition of the environment causes a measurable change in the resonance angle rpduced by the light refracting off the gold-coated prism. A solution containing proteins that might interact with the immobilized bait protein attached to the film is allowed to flow past the biosensor surface. Interacting proteins bind to the bait causing a change in the resonance angle of the light which is measured by a sensor
Which specialised sequence within chromosomes is the DNA sequence located at the ends of linear chromosomes and maintain chromosomal integrity
Telomeres
How do kinesins transport organelles along the microtubules
Both kinesin heads initially contain tightly bound ADP, and move randomly. When one of the kinesin heads encounters a microtubule, it binds tightly. Microtubule binding causes ADP release from the attached head. ATP then rapidly enters the empty nucleotide binding site which triggers the neck linker to zipper onto the catalytic core. This action throws the second head forward, and brings it near the next binding site on the microtubule. The attached trailing head then hydrolyses the bound ATP, releasing a phosphate. As the neck linker unzippers from the trailing head, the leading head exchanges its nucleotide and zippers its neck linker onto the catalytic core, and the cycle repeats.
Which class of molecular chaperone acts directly on the proteins as they leave the ribosome and bind to hydrophobic residues
Hsp 70 class
State the general equation for the addition of a nucleotide to the grown polynucleotide chain
dNTP + (dNMP)n –> (dNMP)n+1 + 2Pi
To which domain does Zn2+ bind in a structural mode
Zinc finger domains
Recall the specific DNA sequence found in telomeres
TTAGGG
Explain how DNA polymerase synthesises the lagging strand
Lagging strand DNA polymerase completes Okazaki fragments in the 5’ to 3’ direction and then starts synthesising a completely new fragment further along towards the 5’ end of the parental template strand
Nucleotide excision repair is only used to repair pyrimidine dimers, T or F
F – it is used to repair a variety of different types of DNA damage including pyrimidine dimers
How can gene replacement be used to study disease
It enables you to test if a present in human patients causes the disease symptoms in mouse by making the same change in the corresponding mouse gene
How do DNA transposons act
Transposase binds to the short simple repeat sequences that flank the transposon itself. These enzymes then induce double-stranded breaks in the DNA sequence to excise the gene from its initial location. This mobile intermediate produced is then able to move to a new location in the chromosome/genome and insert at a random locus
Roughly how long does it take to sequence a human genome now
56 hours
Describe the process of ion exchange chromatography
Diethylaminoethyl (DEAE) beads are 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. 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.
Describe the synthesis of the 5’ cap in eukaryotes
The 5’ cap is present in all eukaryotic mature mRNAs. Its consists of a methyl guanosine trisphosphate cap added to the first nucleotide. The 5’-5’ linkage is unusual and provides stability to the mRNA. The 5’ cap is also required for binding of the eukaryotic initiation factors. The 5’ cap is added when the mRNA is around 20-40 nucleotides long and begins to emerge from RNA polymerase
What is meant when referring to ? helices as heptad structures
7 amino acids for every two turns of the helix
What does homologous recombination rely on
Using the intact DNA sequence information in the undamaged homologous chromosome
What are the main disadvantages of shotgun sequencing
Because of the random sequences produced it requires the sequencing of more the 6x the size of the genome and is hence very inefficient. It will lead to the sequencing of some regions multiple times and there are always gaps. These gaps require progressive sequencing to be filled in.
Give an example of a triple-coiled-coil structured protein and its role in the body
A triple-coiled-coil is the major structural theme in fibrinogen, a protein involved in blood clotting. The fibrous nature of this protein is related to its ability to form blood clots
Describe the basic structure of a zinc finger domain and how it interacts with DNA
Zinc finger domains consist of an ? helix and 2 ? strands. They interact with the major groove of DNA and via guanylyl bases
What is contained in the pellet after the first centrifugation step in differential centrifugation
Whole cells, nuclei and cytoskeleton
How often to mammalian cells divide on average
Every 24 hours
Explain how the enzyme catalyses addition of an amino acid to the tRNA molecule
The aminoacyl-tRNA synthetase first primes the amino acid by the addition of an AMP to the C-terminus. It then uses the adenylated amino acid to form the aminoacyl tRNA
What are the major disadvantages of NMR
Expensive and is limited to proteins under 50kDa and so can often only be used on protein subdomains. Also a very high sample concentration is required as well as the incorporation of the isotope label.
What are the main two types of mutations in the coding sequence
Missense mutations which are characterised by the substitution of an amino acid. Nonsense mutations create premature stop codons which result in a truncated protein
Give some examples of transposable DNA elements
Ac-Dc in maize, P-elements from flies and Tn3/Tn10 from E.coli
What is meant by the transcriptome
All genes expressed by a cell or tissue
Cyclin dependent kinases are important in the temporal control of DNA replication. During which stage of the cell cycle are cdk levels particularly high and particularly low
G1 phase – low cdk activity. S phase – high cdk activity
What is meant by the A site of the ribosome
Aminoacyl tRNA/activation site
Histone methyltransferases mono, di or trimethylated which amino acids within the histone tails
Lysine and arginine
Where is protein methylation often seen and which enzymes and amino acids are involved
Methylation of lysine and arginine residues is often seen in histones and are involved in epigenetics. Methyltransferase enzymes are responsible for this process
What is meant by wobble base pairing and what does this achieve
Wobble bases occur at position 3 in the anticodon and allow the same anticodon to bind to more than one codon
The kinetochores consist of out and inner plate proteins, explain the different roles of these constituent parts
The kinetochore inner plates bind to alpha-satellite DNA and specific centromeric histones. The kinetochore outer plate proteins bind to protein components of the mitotic spindles such as microtubules
Microarrays are an ideal way of comparing gene expression in diseased cells/tissues with the healthy states, T or F
T
Epigenetic modifications facilitate stable changes in gene expression which may persist or the life of the cell of organism but are erased in the germ line, T or F
F – whilst the statement is true, epigenetic modifications are transferred to the progeny these cells can then decide whether to remove the epigenetic change
How can CD be used to infer the stability of a protein upon heating
Less stable proteins will lose its characteristic CD spectrum upon heating
Explain how H3K27 and H3K9 leads to the formation of a Barr body
Lysine 9 and 27 methylation along the entire length of the X chromosome leads to the entire silencing of the chromosome and its shunting to the periphery of the nucleus. This leads to the production of a Barr Body, a highly condensed inactive X chromosome at the periphery of the nucleus of every female somatic cell
What process is used to separate restriction fragments once the DNA has been cut
Gel electrophoresis
By what bonding do side chains of the helices of leucine zippers directly contact the DNA bases
Via hydrogen bonding
Coupled mass spectrometry is usually carried out after affinity chromatography, T or F
F – it’s usually carried out after 2D gel electrophoresis
Specific transcription factors bind close to the promoter region, T or F
F – they bind far away
What is meant by PH domains and what is their function
Pleckstrin homology (PH) domains are involved in membrane binding with functions in signalling and the anchoring of proteins into the membrane
How are mosaic mice then used to produce homozygous knockout mice
Some of the transformed ES cells in the embryo will hopefully have given rise to cells in the gonads. Thus, breeding mosaic animals together will create non-mosaic carries of the transgene in the second-generation. These carriers can then be bred together to produce homozygous mutant animals in the third-generation
How does SH3 bind its ligand
Via aromatic amino acid stacking
? helices are formed by hydrogen bonding within the polypeptide chain, known as intra-chain hydrogen bonding, T or F
T
Describe the process of creating a construct to knockout a gene in a mice model
First, a genomic clone of the gene is obtained and a selection marker is inserted. The selection marker is usually an antibiotic resistance gene such as NEO which encodes for resistance to neomycin. This selection marker is inserted directly into an exon of the gene hence destroying its action. Outside of the exon another selection marker gene is inserted into the construct, this is known as TK. This creates a construct containing two homologous arms of the target gene that flank the NEO gene and a downstream TK gene.
What is the difference between normal nucleotides used in DNA synthesis and those used in dideoxy terminator sequencing
Whereas deoxynucleotide trisphosphates contain a 3’ C-OH bond, dideoxy nucleotide trisphosphates have a 3’ C-H bond which DNA polymerase cannot act on
What is meant by the probability of transcription
There are usually many inputs that alter gene expression, referred to as genetic switches. Each switch responds to intrinsic or extrinsic regulation. The combination of enhancers, repressors, silencers and activators is what determines the probability of transcription.
Microarrays allow comparison of the genomes of different tissues, T or F
F – it allows the comparison of the transcriptomes
What are the implication of translation in the absence of EF-1
There are more errors in the protein sequence
What is the name given to the machinery that carries our RNA splicing and what does it consist of
Spliceosome – a nuclear complex made up of about 150 proteins and 5 RNAs
What does SDS-PAGE stand for
Sodium Diethyl Sulphate Poly-Acrylamide Gel Electrophoresis
Give an example of protein that contains EF-hand domains and describes its role
Calmodulin kinase – requires Ca2+ binding for its structure and regulation. Calmodulin is formed of a single polypeptide chain and contains N and C-terminal globular domains separated by an extended central helix. Each globular domain contains two high-affinity Ca2+ binding sites. Binding of four Ca2+ ions induces major allosteric changes in calmodulin. The two globular heads rotate relative to eachother enabling calmodulin to bind to target proteins and regulate their activity
Explain the process of DNAse I footprinting in the identification of DNA binding proteins
Once you have a known sequence of DNA that you want to discover interacting proteins for, you radioactively label it with 32P. This sequence is then mixed with a cell extract or purified proteins to allow binding of potentially interacting partners. The DNAse enzyme is then added to the mixture to partially digest the sequences by cleaving each piece only once. The sample is then heated to destroy the DNAse enzyme and release the binding proteins. The sample is then run by gel electrophoresis. If a DNA binding protein has bound, then the region of DNA where its binds will be protected from the cleavage by the DNAse enzyme. This will be represented as a missing band in the gel.
More complex organisms have more protein coding genes and less non-protein-coding DNA, T or F
F – they contain more protein and non-protein encoding genes
How many ways can the Double Holliday Junction be resolved
2
Why do proteins fold of their own accord
Proteins often contain hydrophobic regions which need to be hidden in the centre of the structure to achieve a low energy state
What are the constituent parts of M phase
Nuclear division – mitosis and cytoplasmic division (cytokinesis)
What is the disadvantage of reverse mouse genetics
It’s an extremely time consuming and expensive process
Describe the structure of a ? sheet
Beta strands are connected laterally by backbone hydrogen bonds
What phenomena is affinity chromatography said to rely on
Tight interactions (enzyme-substrate binding)
What is the name of the enzyme responsible for catalysing the conversion of sugar(s) into ethanol and carbon dioxide often used in wine making
Zymase
What is the significance of DNA polymerases inability to work de novo
RNA primers required at the start of the replication of the leading strand and at the start of each Okazaki fragment of the lagging strand
What is cell-free mitosis
Technique that allows you to observe mitosis/nuclear division in a test tube outside of the cell membrane. This can be used to study changes i.e. in protein phosphorylation over time after cytoplasmic depletion using antibodies
Whilst in yeast, autonomously replicating sequences (ARS) direct DNA replication initiation, similar structures in humans have proven elusive but seem to be defined by chromatin structure rather than DNA sequence, T or F
T
Coiled-coils are often found in soluble proteins, T or F
F – they are often found in elongated, fibrous proteins
What is the role of RNA polymerase I
Transcribes rRNA genes (28S, 18S and 5.8S)
Which enzyme works with ribonuclease H to repair gaps in the DNA sequence
DNA polymerase – extends across the gaps created by RNA primer removal by ribonuclease H
Due to the fact that DNA synthesis can only occur in a 5’–>3’ direction, how many polymerase enzymes are required per replication fork
2
What are the five main functions of chromatin
Packaging and unfolding DNA in the nucleus, control of DNA replication, repair and recombination, maintenance of chromosome integrity, governing chromosome segregation and the regulation of gene expression
What is different between the synthesis of the leading and lagging strand during DNA replication
The leading strand is synthesised continuously and precedes the synthesis of the lagging strand. The lagging strand however, is synthesised discontinuously i.e. with breaks in the polynucleotide chain
What is the name given to the sections of DNA produced by restriction enzymes
Restriction fragments
What is meant by ribosomes being referred to as ribozymes
They are RNAs that act more like proteins/enzymes by catalysing reactions
Polymerase action causes an uncoiling of the DNA that provides a force on the upstream DNA, how is this tension relived
Topoisomerases release the tension by either making single or double stranded breaks in the DNA upstream hence removing some of the coils from the superhelix
Describe the protein-fraction graph produced in affinity chromatography
Upon addition of the cytosol to the column there is an initial large peak in the relative protein amount which decays away as fraction numb increases. Addition of an eluting substance then causes another peak in relative protein amount due to elution of the bound protein from its substrate
How does codon-anticodon complementation impact GTP hydrolysis by EF-1
The hydrolysis of GTP by EF-1 occurs more rapidly if the codon and anticodon are correctly matched
What is the purpose of the G2/M checkpoint
Checks to see is all DNA has replicated and if the environment is favourable before assembling the mitotic machinery
What is the role of tRNA
tRNA participates in translation where its bound amino acid is added to the growing polypeptide chain when the tRNAs anticodon loop binds to the complimentary codon on the mature mRNA strand
How does competitive ELISA differ from other enzyme-linked immunosorbent techniques
A labelled antibody complimentary to the target protein binds to the antigen. Then another antibody is introduced that competes for binding to the primary antibody. An enzymatic reaction between the antibodies gives a colour
In the reaction catalysed by DNA ligase, two phosphates are removed from ATP to form AMP which then binds to the 5’ phosphate in the polynucleotide chain, the two phosphates are referred to as pyrophosphate, T or F
T
How are specific fragments then isolated once separated and identified
Specific bands are cut out from the gel using a razor and then the DNA contained within it can be purified out
Usually the consensus sequence is the sequence with the highest affinity for the DNA binding protein, T or F
F – often it isn’t
How far apart can ribosomes bind to RNA sequences
80 base pairs
What is meant by there being three possible reading frames
Within a codon there are three different points which can act as different starts points and determine different amino acid sequences
As well as being involved in linking signalling components, what other role does SH3 have
Also as a structural role in maintaining multiprotein complexes
How are the cells that arise from gametes that contain an extra chromosome or missing homologue referred to
Aneuploid
Give an example of a gene involved in homologous recombination that when mutated can cause cancer
BCRA2 – causing breast, ovarian and prostate cancer
How does methylation and acetylation of histone influence transcription
Acetylation and methylation marks in general create binding sites for transcription factors
Transformation of bacteria is an ineffective process, how is this overcome
Integrated into the recombinant plasmid vector is a gene for antibiotic resistance. Once bacteria have been exposed to the vector they are grown on a medium containing that antibiotic. Bacteria that have taken up the plasmid will be the only ones to grow on the medium and thus will contain the target DNA sequence along with the antibiotic resistance gene.
How does DNA helicase separate parental DNA strands at the replication fork
DNA Helicase sits like a nut on a bolt and uses the energy released by ATP hydrolysis to drive a rotational energy which is translated to a force that opens up the replication fork
If a protein contains a hydrophobic centre, what is it likely to be
Soluble
Other than progressive sequencing, what other method has been used to sequence DNA fragments greater than 1kbps and how does this work
Shotgun sequencing. First, a genomic plasmid library is created. The ends clone present in the vector plasmids are then sequenced at random using primers for the vectors. This produces short random sequences from each plasmid that are then assembled by a computer program stitching overlapping regions together to produce a contig. This contig represents the assembled sequence of overlapping regions.
What are the two different types of yeast models
Fission yeast and budding yeast
What attributes of DNA replication make it irreversible
The formation of a new phosphodiester bond and the production of pyrophosphate is coupled to a second reaction catalysed by pyrophosphatase that converts pyrophosphate to two molecules of inorganic phosphate (2Pi)
What are Cdks and how do they act
Cyclin dependant kinases (Cdks) are kinases that phosphorylate proteins involved in specific stages of the cell cycle
What is the approximate size Eukaryotic primers and how often do they occur in the lagging strand
10 nucleotides long, occurring every 100-200 nucleotides in the lagging strand
Enhancer of zeste is one enzyme that methylates a lysine residue. What position in the polypeptide chain does it act
EZH2 methylates lysine 27
What are the different domains of the src-tyrosine kinase
Large kinase domain, small kinase domain, SH2 and SH3
Duplication and multimersiation occurs often in proteins, T or F
T
Explain how zinc finger domains interact with DNA
Zinc fingers consist of an ? helix and a ? sheet region linked together. The ? helical region rests in the major groove of the DNA with amino acid side chains that interact directly with DNA bases via hydrogen bonds. The identity of these interacting side chain amino acids in the helical region of the domain determines which bases it interacts with.
How can this defective recombination pathway seen in some cancers be harnessed as a cancer treatment
Combined inactivation of homologous recombination by mutations and the inhibition of base or nucleotide excision repair by anticancer drugs make cells that suffer DNA damage unable to make adequate repairs to their DNA and die. This is known as synthetic lethality.
How do topoisomerases act
Topoisomerases release the tension created in the polynucleotide chains created by unwinding of the two ssDNA strands. This is achieved by the selective nicking and resealing of regions in the DNA molecule
