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
