DNA and Cancer Flashcards
Which growth factor receptor does masitinib target?
c-Kit
Which cancers are common in golden retrievers and rottweilers?
Lymphoma and osteosarcoma
What is astrocytoma a cancer of?
Glial cells in brain
What are the three cell extrinsic hallmarks of cancer?
Angiogenesis, metatatic potential, evasion of immune destruction
How do cancer cells evade immune destruction?
Produces cytokines to dampen immune response
What are the six cell intrinsic hallmarks of cancer?
Growth signal autonomy, resistance to inhibitory growth signals, unlimited replicated capacity, reprogrammed cell metabolism, resistance to apoptosis, genetic instability
Why do cancer cells have growth signal autonomy?
Cell can’t recognise them so not dependent on them
What is Warburg Metabolism?
Where tumour cells use lots of glucose, use it to produce lactate instead of oxy phos and use the remaining carbon for raw materials.
What are the angiogenic activators?
VEGF and PDGF
What are the angiogenic inhibitors?
Angiostatin and endostatin
What is a common metastasis site?
The perivascular cuff
What does Vascular Endothelial Growth Factor do?
Binds receptors on endothelial cells lining blood vessels, stimulates them to secrete matrix metallo-proteinases to degrade extracellular matrix and allow tissue remodelling so the activated endothelial cells mirgate towards the tumour
Where is platelet-derived growth factor secreted from and what does it do?
From activated endothelial cells, it is a chemoattractant for smooth muscle cells
What drug is an antibody for VEGF and what are its side effects?
Avastin, causes aneurysms and perforations
How does the primary tumour produce angiostatin and what are its effects on the secondary tumours?
By cleavage of plasminogen, stops angiogenesis and metastasis
Which part of the DNA do alkylating agents and aromatic amines react with and what does this form?
Nucelphilic sites on purine and pyrimidine rings, forms DNA adduct (covalent bond with carcinogen)
What is the Ames test?
Measures ability of chemical to mutate the Salmonella genome - grow bacteria that can’t synthesize His in the absence of His and only mutated colonies will grow
What are the five types of DNA repair?
One-step repair, nucleotide excision repair, base excision repair, recombination repair (HR or NHEJ) or mismatch repair
Which enzyme does one step repair use?
Alkyltransferases to repair damage from alkylating agents
What kind of defect does nucleotide excision repair detect?
Helix-distorting lesions
What are some epigenetic changes which may cause cancer?
Methylation of tumour suppressor transcriptional promoter sequences, post-translational modification changing stability or activity, non-coding microRNAs inhibiting translation of proteins involved in cancer
What are the effects of increased microRNA in a tumour and in normal tissue?
In tumour is acting as oncogene (inhibiting suppressor), if in normal tissue it’s acting as tumour suppressor (inhibiting oncogene)
Which part of the DNA does ionisation affect?
The base
What does deamination of cytosine produce?
Thymine
What does the oxidative pentose phosphate pathway produce?
Nucleic acids
What two metals encourage benzene to bind to DNA?
Cobalt and nickel
What does plasminogen cleavage produce?
Plasminogen >Plasmin > Angiostatin
What do macrophages secrete?
Growth factors
What is sis?
A growth factor
What is erbB?
A growth factor receptor
What is the point of the strep experiment?
To show that DNA (not protein or lipid) in the “transforming principle” transferring pathogenic properties
What’s the process of the strep experiment?
S strain is pathogenic because it has a smooth coat to protect it from the immune system. Fraction the S cells into components, test which transforms R to S.
What’s the point of the blender experiment?
To show that genetic material used by viruses is DNA
What’s a bacteriophage?
A virus which infects bacteria
What’s the process of the blender experiment?
Radioactively label either the DNA or the proteins of the virus and add them to the bacteria they infect. Use a blender to separate bacteria and viruses and then centrifuge. Look where the radioactivity goes - protein stays within the phage, DNA moves to the bacteria
Which atoms are labelled in the blender experiment?
32P in the DNA, 35S in the methionine of the proteins
What are the two layers in the test tube called after centrifuging in the blender experiment?
The pellet at the bottom (bacteria) and the supernatant (the phage)
What’s the structure of the backbone in terms of sugar and phosphates?
Alternates sugar and phosphate
What does the base attach to?
The sugar
Which carbon of the sugar do the base and the phosphate attach to?
Sugar is C1, phospahte is C5.
What is at the 5’ end and what is at the 3’ end?
Phosphate at 5’, OH at 3’
What’s the bond called in DNA between the carbons?
Phosphodiester
Base + ribose = ?
Nucleoside
Nucleoside + phosphate = ?
Nucleotide
How were Chargaff’s rules worked out?
Heat to break phosphodiester bond, use phosphodiesterase to get nucleotides, use chromatography and measure intensity of each band
Why can RNA form a double helix or fold on itself?
2’ hydroxyl
What are the three forms of the double helix?
B (normal), A (RNA double helix) and Z (left handed)
What does the A form of the double helix look like?
Wider, shallower minor groove
What are some characteristics of the DNA double helix?
Right-handed, phosphates repel each other, planes of bases perpendicular to vertical axis, one turn is 10.5 base pairs, rises 34A per turn
What area of the cell are chromosomes found in?
Chromosome territories
What is a nucleosome core particle?
Loop of DNA wrapped around a histone
Which protein can wrap DNA around proteins?
Integration host factor
Which proteins move nucleosomes? What does this affect?
Remodelling proteins using ATP, affects availability for transcription factors
What is supercoiling?
Over/underwinding of a strand which introduces tension so wraps around itself
What do topoisomerases do?
Change degree of supercoiling by cleaning and rejoining DNA strands
What is a nucleoid?
A rosette of DNA made by domains of independent supercoiling
Which are the four subunits found in a histone octamer? Which subunit packs the nucleosomes together?
H2A, H2B, H3 and H4. H1 packs together.
Which amino acid is positively charged in histones?
Lysine
Does methylation or acetylation of histones repress or acetylate transcription?
Methylation represses, acetylation in promoter regions
What is conservative replication?
Would reform original strand?
What is dispersive replication?
Produces strands with fragments of parent and daughter DNA
What is steric exclusion?
Where replicated helicases form hexameric rings where one strand is threaded through and the other is peeled off
What are some properties of the origin of replication (OriC)?
AT-rich, one per chromosome, has 5 DnaA boxes
What does DnaA do?
Bind, interact and partially melt the double helix, act as loading site for DnaB
What does DnaB do?
It’s a helicase which unwinds the double-stranded DNA
What does DnaC do?
Brings the DnaB
What does DnaG do?
It’s a primase which makes a short RNA primase
Which DNA polymerase replicates the leading and lagging strand in prokaryotes?
DNA pol III
Which DNA polymerase in prokaryotes has 5’-3’ exonuclease activity to remove the RNA primer?
DNA pol I
Which DNA polymerase has a B clamp dimer and what does this do?
DNA pol III, holds it onto template so it’s capable of progressive synthesis
Which complex loads the B clamp dimer?
Gamma complex
What is a holoenzyme complex?
In bacterial DNA replication leading and lagging strand synthesis is coupled instead of going in opposite directions
What is the trombone model?
Lagging strand template is primed but bent back around to form a loop which is periodically released in bacterial DNA replication
What are type I topoisomerases for?
Nick one strand, pass it around the other, reseal. Removes supercoils in front of the replication fork
What are type II topoisomerases for?
Cuts both strands if two replication forks collide
Why do topoisomerases use covalent bonds with the DNA?
Preserves bond energy, ensures DNA molecules aren’t released
In eukaryotes, what recognises the origins of replication?
Origin recognition complex (ORC)
What does ORC recruit and what does it need to do this?
MCM helicase, needs Cdc6 and Cdt1
What do eukaryotes use instead of SSB?
RPA
Which primase is used in eukaryotes?
PriL, PriS, B subunit, DNA pol alpha complex
Which DNA polymerase is used in eukaryotes?
DNA pol alpha, beta, E
What is the sliding clamp in eukaryotes?
PCNA
What is the clamp loader in eukaryotes?
RFC
What co-ordinates primer removal in eukaryotes?
Coordinated by PCNA
What does RNaseH do in eukaryotes?
Removes the RNA primer base paired to the DNA
What does Flap Endonuclease Fen1 do in eukaryotes?
Removes the flap that DNA creates when it displaces the primer
How is the primer removed in eukaryotes?
Primer removed by RNaseH, DNA polymerase displaces it creating a flap which is removed by flap endonuclease Fen1
What is the aim of the genome-wide association study?
Identify alleles relating to a disease
Which chromosome doesn’t have crossing over?
Y
What are some characteristics of euchromatin?
Loosely packed, at chromosome arms, gene rich, replicated in S phase, undergoes recombination, transcribed
What are some characteristics of heterochromatin?
Densely packed, at centromeres and telomeres, gene-poor, replicated late S phase, repetetive, no recombination, not transcribed
What is a telocentric chromosome?
Centromere is at the end of each chromosome arm
Why are telomeres needed?
Because lagging strand either wouldn’t be replicated or would be left with DNA-RNA duplex causing loss of genetic information
What’s the telomere sequence in humans?
TTAGGG
What is the structure of telomerase?
Has protein and RNA component, is a self-templating reverse transcriptase
What are T and D loops?
Found in telomeres, T loops are where unpaired end folds back and closes off, D loop is where one strand is displaced so the T loop can bind
Which nucleotides did Sanger use for chain termination?
Dideoxynucleotides (ddNTPs) which terminates chain as no 3’OH group
In euchromatin which repeats are not derived from transposons?
Simple repeats eg CACACACA and segmental duplications
In euchromatin which repeats are derived from transposons?
Transposon-derived repeats and inactive copies of partially retrotransposed cellular genes
What are the two types of mobile genetic elements?
Transposons via transposase or retrotransposons which go DNA>RNA>DNA then inserted back
What are long/short interspersed nuclear elements, retrovirus-like elements and DNA transposon fossils types of?
Mobile genetic elements
What is FISH used to look at?
Chromosomal rearrangements and gene copy numbers in cancer cells
What is the bacterial restriction-modification system?
Methylate their own DNA at adenine residues using DNA adenine methyltransferases which makes it different to viral DNA
Where do type II restriction endonucleases cut?
At palindromic sequences
What are the two types of type II restriction endonucleases?
EcoRV produces blunt ends, EcoRI produces sticky ends
Which dye is used when looking at DNA?
Ethidium bromide which intercalates into the DNA and fluoresces orange under UV light
Which “blots” are for what?
Southern for DNA, Northern for RNA, Western for protein
How do you purify plasmids from bacterial genomic DNA?
Alkaline lysis
What are the temperatures for melting, annealing and extension of DNA?
90, 55-60 and 72
What do you need for PCR?
Oligonucleotide primers, template DNA, dNTPs, buffer and Taq polymerase
What are the three endogenous causes of DNA legions?
ROS, reactive chemicals (S-adenosylmethionine) and chemical instability (deamination/depurination)
What are the four types of DNA damage?
Transition, transversion, alteration to backbone or loss of covalent identity of backbone
What is a transition and what is a transversion?
Transition is purine>purine and transversion is purine>pyrimidine
What does UV light cause?
Thymidine dimers through a cyclobutyl ring
Which enzymes converts a thymidine dimer back?
DNA photolyase
What does 8oxoG cause and which enzyme fixes it?
GC>TA transversion, removed by DNA glycosylase
Which chromosome is the Huntingtin gene on?
4
Which codon expansion causes Huntington’s diseases and what does it code for?
CAG for glutamine
How do the hairpins cause triplet expansion is Huntington’s disease?
Replication machinery may accidentally expands it or repair machinery may expand it while trying to fix it, could be slipped-strand intermediates if one strand has more repeats than the other, CNG (C-nucleotide-G) departs from normal B form DNA
Which enzymes converts the superoxide anion to peroxide?
Superoxide dismutase
What is the reaction called which converts peroxide and Fe2+ to OH radical, OH- and Fe3+?
Fenton
What can cause C>U?
Can be spontaneous, treatment with nitrites or via oxidative conversion
How is C>U transversion fixed?
Uracil DNA glycosylase recognises, flips and cleaves the glycosidic bond. A nucelease cleaves the backbone at the abasic site, acts as priming site for DNA pol I or b, puts C back opposite templated G
What does DAM methyltransferase do?
Methylates A in GATC sequence in bacteria to protect from restriction endonucleases
What does DNA methyltransferase do?
Methylates C in eukaryotes
In which areas is methylation common?
CpG islands
What does methylation do in CpG islands?
Switch off gene expression, responsible for genomic imprinting
How is Prader-Willi syndrome caused?
Paternal is deleted, maternal is silenced
How are thymidine dimers repaired?
Detected, DNA around lesion unwound, single-stranded gap produced, polymerase fills it in, ligase sticks it
What do mutations in NER cause?
Xeroderma pigmentosum
Which cell phases is NHEJ important in?
G1 and G0
What is it called when the ends are lost during NHEJ?
synapsis
How can bacteria tell which end is the template?
DNA is usually methylated but is only hemimethylated when first produced
What is the process of DNA mismatch repair?
Strand flanking error is excised and polymerase fills it in
What cancer does mutations in DNA mismatch repair cause?
Colorectal cancer
What does homologous recombination use as a template?
The other copy of the chromosome
Which enzymes in prokaryotes and eukaryotes scan the genome for the identical double-stranded sequence?
RecA (prok) or RAD51 (euk)
Which complex slides Holliday junctions in bacteria?
RuvAB
What’s the process of homologous recombination?
3’ tail created, D loop forms in template chromosome, ends join
Which protein does BRCA2 control activity of?
HR protein Rad51
What does translocation between chromosomes 9 and 22 cause?
Chronic myelogenous leukaemia
Which enzymes add extra nucelotides to increase variation in antibodies?
Terminal transferase
What happens to pyrophosphates released during RNA synthesis?
Hydrolysed to phosphate via pyrophosphatase
Why is prokaryote RNA polymerase so error prone?
No 3’-5’ exonuclease or correction mechanisms
What’s the difference between the core enzymes and holoenzyme of RNA polymerase?
Holoenzyme has sigma subunit as well as alpha2, beta and beta’
What does the sigma subunit do?
Holds machinery onto DNA, different one for different promoters, suppresses random binding
Why are promoter sequences asymmetric?
So RNA polymerase will only bind in one direction
How is synthesis initiated in prokaryotes?
Holoenzyme binds to promoter, still closed complex, then the two DNA strands open to form open complex, correct NTP is selected and incorporated and the pyrophosphate is lost, moves on by one nucleotide, sigma lost after 6-8 nucleotides to initiate synthesis of another chain
What are the promoters in prokaryotes?
-35 and -10 (Pribnow box)
What is an operon?
A promoter controlling a number of genes
What is a polycistronic transcript?
mRNA which encodes several polypeptides
Why does a run of 6U terminate synthesis?
Doesn’t bind to DNA so it falls off
How do sigma factors respond to environmental conditions?
Recognise promoters of genes where transcription is induced due to an environmental condition eg. heat shock
What is rho independent termination?
Terminates due to GC hairpin and 6U in RNA where palindrome forms stable, GC-rich hairpin so RNA polymerase pauses and then dissociates
What is rho dependent termination?
Have hairpin but no U tract, preceded by high Cs, rho (a helicase) binds to the C rich RNA and unwinds the DNA-RNA duplex while it’s paused
How does CAP affect RNA polymerase activity?
Stabilises it (a positive regulator)
What induces synthesis of B-galactosidase?
B-galactosides
How does alpha-amanitin work?
Interacts with RNA pol II and blocks transcription
How does Actinomycin D block RNA synthesis?
Binds to dsDNA by intercalating between neighbouring GC pairs, stops DNA and RNA polymerase accessing it
How does rifamycin work?
An antibiotic - binds the B subunit of RNA polymerase and doesn’t affect eukaryotes, blocks chain elongation
What does microRNA do?
Regulates mRNA
Where is the TAT box and which RNA pol does it promote?
-25
Why does RNA pol II need general transcription factors?
Will use either DNA strand
What are the general transcription factors of RNA pol II?
TFIIA TFIIB TFIID TFIIE TFIIF TFIIH
What are the three types of RNA polymerase in eukaryotes?
I - make 45S pre-rRNA, II makes pre-mRNA and U1-U5 snRNAs and miRNAs, III - other small RNAs e.g. tRNA
How many subunits does RNA polymerase have?
12
Which amino acids are transcription factors rich in?
Acidic, glutamine or proline
What do transcription factors bind to?
Upstream promoter elements
What do the two domains of the trainscription factors bind to?
One for DNA, one for co-activators or co-repressors
How do you identify functional parts of promoters?
Choose an easily assayable enzyme eg firefly luciferase and put it in a plasmid, put potential regulatory fragments upstream and see if it’s expressed, keep removing parts and you’re left with the bit the TFs bind to
How do you do DNA footprinting?
Label some DNA with 32P, divide it into two tubes, add binding proteins to one tube, break it up using DNAse I, compare both using electophoresis and there will be a gap at the binding site
What is the gap at the binding site called in DNA footprinting?
Protected region
Which enzyme is used to break up DNA using footprinting?
DNAse I
What forms a pre-initiation complex and what does it need to initiate transcription?
Transription factors and RNA pol II
What makes up TFIID?
TATA + TATA binding protein + TBP associated factors
What initiates PIC assembly? And how?
TATA binding protein by bending DNA so other proteins can bind
What does TFIIF bring?
RNA polymerase II
What is the order of promoter elements from upstream to downstream?
Enhancer > response elements > Upstream promoter elements > Promoter core elements > Transcription start
What happens to nucleosomes during transcription?
Don’t dissassemble but most promoters are nucleosome-free
What do AP1/AP2 do?
Enhancers recognised by c-Fos and c-Jun.
What are GRE and CRE?
Glucocorticoids and cAMP response elements
What does CREB-binding protein do?
It is a HAT
Where are histone deacetylases found?
Repressor transcription factors
What does ChIP do?
Finds direct evidence for binding sites of transcription factors
What is the process of ChIP?
Use formaldehyde to crosslink chromatin and the binding proteins, shear DNA into small fragments using ultrasound, use an antibody to the TF to fish out the TF and the DNA it binds to, use a protease so only the DNA is left, identify the DNA using PCR
How does transcription rate enhancement occur when enhancers are far away from promoters?
DNA bending
Why are many TFs dimers?
Response elements are symmetrical
What are some tissue-specific transcription factors?
MyoD in myoblasts and Oct-2 in light and heavy immunoglobulin genes in B lymphocytes
What do Hox genes encode?
Transcription factors
What is the homeodomain of a Hox gene?
Binds specific promoter sequences in genes encoding segment identity
How does CREB activate pol II?
adrenaline/glucagon > adenyl cyclase > cAMP > PKA > phosphorylates CREB > binds CBP (HAT) > which activates pol II
What is CREB a transcription factor for?
cAMP response element (CRE)
How do steroid/thyroxine/vitamin D transcription factors work?
Hormone binds to receptor, receptor activated, binds to hormone response elements, transcription activated
What are the mechanisms of action of the glucocorticoid receptor?
Cortisol binds, hsp90 dissociates, receptor dimerises, moves into nucleus. ALSO: cortisol binds, transcriptional activator domain activated, activates gene expression following DNA binding
What happens to chromtin structure after a ligand binds to it?
Receptor alters chromatin structure so general transcription factors can access core promoter elements
What makes up AP1?
c-Fos + c-Jun
What has to happen to p53 for it to activate transcription?
Phosphorylated by a CDK
What causes retinoblastoma?
Loss of tumour suppressor Rb - it’s phosphorylated to allow transcription and if this is inappropriate you get retinoblastomas
What is the structure of the eukaryote and prokaryote cap?
Eukaryote: m7G5’ppp5’N and Prokaryote: p5’N
What is capped pre-RNA bound by?
RNPs for stability
What are the roles of the 5’ cap?
Protect from 5’ exonucleases, increases splicing efficiency of 5’ proximal introns, export to cytoplasm and translation initiation
What are the roles of the poly(A) tail?
Protects from 3’ exonucleases, controls degradation rate and enhances translation rate
Where is the poly(A) tail added and what happens to it during transport to the cytoplasm?
Added in the nucleus then shortens
How is the eukaryotic cap added?
Phosphate removed from 5’pppN end of new RNA, enzyme adds GMP from GTP, forms G5’ppp5’N, the added G is methylated at the 7 position, the second methyl group is added to the 2’OH of the first ribose
What is the methyl donor for the added G in the eukaryotic cap?
S-adenosylmethionine
Why is the G5’ppp5’N unusual?
5’-5’ bond
What binds to accessory factors and co-ordinates adding of protein-coated As?
CPSF
What do histone genes not have?
Introns
Why is the precise 3’ end with the poly (A) tail needed?
Pol II transcription doesn’t terminate precisely
What is the process of adding the poly(A) tail?
Pre-mRNA is first cleaved between AUAAA and GU/U-rich sequence, then poly(A) polymerase adds As, then the downstream fragment is degraded
Which enzyme adds the poly(A) tail?
Poly(A) polymerase
What are genomic DNA libraries for?
To determine gene structure, characterise sequences regulating transcription, express bacterial proteins
What are cDNA libraries for?
To determine mRNA sequence, express eukaryotic proteins, characterise regulatory sequence in UTR
How do you find which mRNAs are found in a specific cell type?
Extract RNA from cells, add reverse transcriptase, and RNase so single strand of DNA is left, use DNA polymerase to make the other strand (called cDNA), clone into vector
Which particles does splicing need?
snRNPs
What are the different types of snRNPs and why are they called this?
U1, U2 etc because uridine-rich
What are snRNPs transcribed from and by what?
Multicopy genes by RNA pol II except U6 which is pol III
What causes lupus?
Antibodies against snRNP proteins
What is the process of splicing?
2’OH of branch point adenosine attacks phosphate at 5’ end forming a lariat and the 5’ exon is released. The 3’OH of the released 5’ exon attacks the phosphate at the 3’ end of the intron. The exons join together and the lariat is released and degraded.
What happens to the number of phosphodiester bonds during splicing?
Conserved in number
What do introns have at the 5’ and 3’ end?
GU at 5’, AG at 3’
Which end of the splice site is the donor site and which is the acceptor site?
5’ is donor, 3’ is acceptor
What does U1 bind to?
The GU
What does U2 bind to?
The AG
What does U2AF bind to?
The pyrimidines behind the AG
What happens if there are mutations in the splice sites?
Either splicing inactivated or cell uses “cryptic splice sites” with similar sequences
What is alternative splicing?
Same gene does different things depending on where it’s spliced to increase coding capacity
What are some ways of alternative splicing?
Different 5’ site, common 3’ site, whether to splice or not, exon skipping, common 5’, different 3’
In which snRNPs does the RNA recognise the sequences at the start of each intron?
U1 and U2
How many snRNPs make up the spliceosome?
5
Which snRNPs make up the catalytic core of the spliceosome?
U2 and U6
What is the major type of RNA editing in eukaryote nuclei?
Base conversion
What is hydrolytic deamination and what changes can it cause?
Where C or A from genome is converted to U or I (inosine), can change codon, introduce stop codon or affect splicing or increase coding capacity
What is poly(A) tail shortening controlled by?
3’ UTR sequences
What does the neurofibromatosis gene encode and how does it work?
Encodes neurofibromin, a tumour suppressor which contains GTPase activating protein which interacts with Ras to regulate signal transduction
What does tumour editing in the NF1 gene cause?
Truncated protein at GAP domain and inactivates tumour suppressor function
What is the pathway for RNA degradation?
Deadenylation, decapping and 5’-3’ exonuclease
What is nonsense-mediated decay?
A degrading mutation involving premature stop codons
What happens to histone mRNA before targeted degradation?
Polyuridylated (gets poly(U) tail)
What do miRNA and RNAi (RISC) do?
Destabilise mRNA
What are the three stop codons?
UGA (amber), UAG (ochre) and UAA (opal)
How do you find out which codons do what?
Use synthetic polynucleotides eg) UUUUUUUU is poly(Phe)
What is inosine?
A modified purine
What is the initiation methionine cleaved by in eukaryotes?
aminopeptidase
Which kinds of methionine are found at the P site and the A site?
Met-tRNAi at the P site and Met-tRNA at the A site
Why isn’t tRNA recognised as harmful dsRNA?
Lots of modified bases
What does the variable arm allow the tRNA to do?
Bind to its specific amino acid
What does the amino acid join to in the tRNA?
A CCA at the 3’ end with a 3’/2’ OH
What is tRNA joined to an amino acid called?
Charged or aminoacyl
Where is the anticodon compared to the bound amino acid?
The opposite end
How are different isoforms of the WT1 gene generated?
Alternative splicing, alternative translation start sites and RNA editing
What do mutation in the WT1 gene cause?
Kidney cancer (Wilms’ tumour), kidney disease or gonadal dysgenesis (male-female dysgenesis)
What does the WT1 protein do?
A transcription factor which recognises GC/TC rich promoter sequences
What is the structure of the WT1 protein?
A two-domain protein with N-terminal proline and glutamine-rich activation domain and C-terminal DNA-binding protein with 4 Zn fingers
How many alternatively spliced variants do normal cells have?
4
What are the mutations in the Wilms’ tumour?
Either in the coding region or introns altering the splicing pattern (can lack DNA binding protein or parts of activation domain) so it no longer functions as a tumour suppressor
What does Frasier syndrome cause?
Affects kidney and gonads and XY has dysgentic gonads
What causes Frasier syndrome?
Intron mutation disrupts alternative splice site forming +KTS isoform which doesn’t bind DNA and functions as an RNA processing factor
Which part of the amino acid joins to the tRNA? What catalyses this?
The carboxyl group - aminoacyl tRNA synthetase
What accounts for the mass and shape of the ribosome?
RNA for mass, protein for shape
What’s the “wobble position”?
The tRNA backbone is flexible so last base is allowed to be different
What are the weights of the bacterial and mammalian large subunit?
Bacterial 50S, mammalian 60S
What are the weights of the bacterial and mammalian small subunit?
Bacterial 30S, mammalian 40S
What is the loading mechanism of tRNA?
The amino acid > enzyme-aminoacyladenylate and pyrophosphate is released, then tRNA > aminoacid-tRNA and AMP is released
What happens if the wrong AA if used during tRNA loading?
The complex falls apart
What is a polyribosome?
Several ribosomes independently translating the same RNA molecule?
Where do ribosomes live?
A pool of free subunits which would just associate into inactive complexes
What happens to ribosomes when protein synthesis is initiated?
Small subunit binds to mRNA and large subunit joins
Why can mRNA be translated as it’s made in bacteria?
Synthesized and translated 5’-3’
What is before the methionine in bacteria?
Formyl
What removes the formyl from the methionine?
deformylase
What removes the methionine from the rest of the protein?
Aminopeptidase
How do bacteria know the correct initiation site?
The Shine-Dalgarno sequence right before the methionine codon which binds to the ribosome
If a gene is polycistronic, how many Shine-Dalgarno sequences are there?
One for each protein
How do eukaryotes know the right initiation site?
Always monocstronic and the right AUG is always the first from the 5’ end
How do eukaryotes scan for the correct initiation site?
The 40S subunit binds to the extreme 5’ end and scans to the 3’ end, when it finds the AUG the Met-tRNA locks on and the 60S subunit joins
How is the 40S subunit recruited?
5’ cap binds the protein complex eIF4F (eukaryotic initiation factor) which recruits the 40S subunit
What is internal initiatiob of translation?
The uncapped, long 5’UTR has lots of AUG so not translated by scanning ribosomes, instead there’s an IRES (internal ribosome entry site) which directs the ribosome to the right entry site
What is a dicistronic assay?
To find an IRES - put a sequence you think may be an IRES between two genes, normally the second one wouldn’t be translated but if the sequence is an IRES then it will
In what situations can the IRES be used?
When cap-dependent translation is reduced eg) mitosis, apoptosis, stress or some viruses like polio, FMV
What binds to the P and A site?
Peptide to the P site, amino acid to the A site
What does the amino acid need to bind to the A site?
Elongation factor and GTP hydrolysis (elongation factor dissociates after)
Which elongation factors are used in bacteria and eukaryotes?
EFTu in bacteria and eEF-1 in eukaryotes
What enzyme does the larger ribosome subunit have?
Peptidyl transferase
Which elongation factors are needed for translocation in bacteria and eukaryotes?
EFG in bacteria, eEF-2 in eukaryotes
How is transcription terminated?
When there’s a stop codon, a release factor binds which modulates peptidyl transferase activity and makes it harder to transfer polypeptide to H2O so it’s released
What does “mycin” mean?
It binds to rRNA
How does streptomycin work? What does it cause at low concs?
Binds to 30S subunit and inhibits initiation, causes mRNA misleading at low conc
What does diptheria toxin do?
Covalently modifies eEF2 (translocation elongation factor) which inhibits translocation
What does ricin do?
Has glucosidase activity which removed a single adenosine from eukaryotic 28S rRNA and inactivated the ribosome
What does tetracycline do?
Inhibits aminoacyl-tRNA binding to 30S subunit
What do oxazolidines do?
Inhibit formation of formylmettRNA-30S ribosomal subunit-mRNA subunit
What are oxazolidines effective against?
Gram-positive
What does chloramphenicol do?
Inhibits peptidyltransferase activity of the 50S subuni
What does erythromysin do?
Binds to 50S subunit at the entrance to the peptide exit tunnel and blocks nascent peptide progression
What are some global modifications to initiation factors by phosphorylation?
eIF2 phosphorylation prevents it from being recyled from its GDP bound to its GTP bound state and (the kinase activity is increased if dsRNA from viral RNA is present)
How is translation of a particular mRNA repressed?
Specific binding of an RNA binding protein to a motif in the 5’ or 3’ UTR
What does ferritin do?
Protects the cell from too much iron by sequestering it
How is ferritin synthesis increased during increased iron levels?
mRNAs encoding it have a hairpin in the 5’ UTR called the iron response element. This is bound by iron regulatory protein to prevent it binding to the 40S ribosome inhibits ferritin translation - affinity is reduced in increased iron
How does microRNA inhibit mRNA expression?
Imperfectly base-pairs with 3’UTR of target mRNA which inhibits translation or degrades the mRNA or the protein
How is siRNA used in research studies?
Eliminates gene expression - by causing “RNA interference” or RNAi by forming a perfect duplex with target RNA causing its degradation
What are riboswitches?
RNA switches in the 5’UTR or introns which physically bind to
What does the tryptophan operon do?
Controlled by tRNAtryp, if not enough then induced pause and RNA structure changes
What are the two processes of bulk destruction by lysosomes?
Chaperone mediated autophagy of proteins containing KFERQ amino acid sequence and macroautophagy
What is the structure of the proteasome?
A cap at each end and a staff made of two outer and two inner rings
How are proteins degraded by the proteasome?
The ubiquitinated protein binds to the proteins in the cap, it’s unravelled and the Ubs are spat out, the protein is fed into the shaft to get to the two inner rings for proteolysis and it’s then degraded into 8 amino acid-long peptides whihc are released?
What does E1 do?
Ubiquitin-activating enzyme
What does E2 do?
Ubiquitin-conjugating enzyme
What does E3 do?
Ub ligase
What’s the process of linking ubiquitin to the lysine of proteins?
E1 uses ATP to covalently link Ub’s C-terminal glycine to its own SH group, E2 takes the Ub from E1 and ligates it to its own SH group, E3 takes the Ub from E2 and ligates it to the lysines in the proteins for destruction, then more and more Ubs are added lysine > glycine
What kind of bond is between to Ub and the lysine?
isopeptide
What are three ways of targeting proteins for destruction?
5 amino acids at N-terminus after Met cause it to be degraded faster, PEST sequences can be phosphorylated on S or T which can aid Ub dependent or independent destruction, or there can be a D box in the N terminus of cyclins which is required for their unbiquitination
What is the order of phases in the cell cycle?
M > G1 > S > G2
What does unphosphorylated Rb bind to and what effect does this have on the cell cycle?
E2F so can’t transcribe gene and can’t progress to late G1
What happens during M phase?
Mitosis then cytokinesis
What does G1/S checkpoint require?
Controls CDK2, requires no damage to DNA structure
What does the G2/M checkpoint check?
Replicated DNA
What are CDKs?
Serine/threonine kinases
What activates CDKs?
Cyclins
What inhibits CDKs?
CKIs such as Ink4 and p21/Kip1
What separates the early and late G1 phase?
Restriction point
What does the early G1 phase require to proceed and what happens if there are none?
Mitogens, exits to G0 phase if none
What does the G1 restriction point require to proceed?
Hyperphosphorylation of Rb by CDK4 and cyclin D
What do CDK4 + 6 respond to?
Growth regulatory signals
What do CDK 4, 6, 2 and 1 promote?
Cell cycle progression
What do CDK 7, 8 and 9 regulate?
Transcription
Which tissues does CDK5 have functions in?
Specialised
How many D cyclins are there in mammals?
D1, D2 and D3
What are the major cyclins and CDKs during G1?
Cyclin D and CDK4/6
What are the major cyclins and CDKs during G1/S?
Cyclin E and CDK2
What are the major cyclins and CDKs during S?
Cyclin A and CDK 1/2.
What are the major cyclins and CDKs during M?
Cyclin B and CDK1?
What phosphorylates CDKs?
CAK and Wee1
What dephosphorylates CDKs?
Cdc25
What activates CDKs?
Suc1
How does p27 and p16 inactivate CDKs?
p27 holds inactive conformation and p16 blocks the cyclin binding site
What are the two classes of CKIs?
Ink4 (p16, p12, p18 and p19) and Cip/Kip (p21, p27, p57)
What do Ink4 CKIs target?
CDK4/6
WHat do Cip/Kip CKIs do?
Interfere with cyclin D/E/A/B-dependent kinase complexes
Which ligases control desruction of regulatory proteins?
APC/C and SCF
What is licensing of DNA replication?
When the MCM helicase is loaded onto the origin
What are the 4 cell cycle checkpoints?
G1/restriction, DNA damage, G2 and spindle assembly
During which phases is MCM loading blocked?
S, G2 and M
Why is MCM loading blocked during G2/M?
Lack of Cdc6/Cdt1 to load MCM helicase
What inhibits Cdt1 and when does it accumulate?
Geminin during S phase
What degrades geminin, why and when?
APC/C in M so it can function again in G1
How is replication initiated at the start of S phase?
MCM4 phosphorylated by DDK which activates MCM helicase, replication complexes fire and phosphorylated lots of proteins (most importantly Sld2 and Sld3 which recruit the pre-initation complex)
What does phosphorylation of ORC and Cdc6 trigger?
Degradation and nuclear export which prevent re-replication for that cell cycle
What do mitogenic signals stimulate?
CDK4/6 and entry into the cell cycle
What to antiproliferative checkpoints inhibit and induce?
Inhibit CDK4/6 and induce p16 expression
What does active CDK4/6 phosphorylate?
RB
What does CDK4/6 initiate and activate?
Initiates transcription and stability of cyclin E and A and activates CDK2
What do p21 and p27 inhibit?
CDK complexes
After replication, what phosphorylates G2 targets?
CDK1-CyCA/B complexes
If there is no DNA damage, what is activated to progress into mitosis?
CDK1-CycB
When/why is cyclin B degraded?
For anaphase and daughter cell production
What are newly replicated sister chromatids held together by?
Cohesin
What links centromeric DNA to spindle microtubules?
Kinetochores
What is bi-orientation?
Each sister chromatid has to attach to microtubules from both poles
What are some CDK4 mutations in cancer?
Mutation in melanoma which blocks Ink4 binding, hyperactive in epithelial malignancies
What are some CDK6 mutations in cancer?
Overexpressed in leukaemia, mutated in mesenchymal tumours because Ink4 and Cyclin D misregulated
What happens to E cyclins in cancer?
Overexpressed
What happens to p21 and p27 in cancer?
Silenced
What are the five stages of cell division?
Interphase, prophase, prometaphase, metaphase, anaphase, telophase
What triggers entry into mitosis?
Mitosis-promoting factor (increase in cyclin B-CDK1)
What are the three key kinases in cell division?
Cdk1, Aurora B, PLK1
Where is the spindle assembly checkpoint and what happens if it’s inactive?
In prometaphase, exit from mitosis occurs
What is Cdk1 activity like during metaphase, anaphase and telophase?
High in metaphase, low during anaphase and inactive during telophase
Where does Aurora B go during metaphase, anaphase and telophase?
Centromeres during metaphase, central spindle during anaphase and the midbody (but inactive) during telophase
Where does PLK1 go during metaphase, anaphase and telophase?
Kinetochores/centrosomes during metaphase, central spindle during anaphase and degraded during telophase
What happens during prophase?
DNA condenses into chromosomes and mitotic spindle develops
What happens during prometaphase?
Nuclear membrane breaks down into vesicles, golgi membrane breaks into vesicles
What happens during metaphase?
Kinetochore assembles and joins microtubule
What happens during anaphase?
Sister chromatids separate, kinetochore microtubules shorten, astral microtubules pull poles apart, interpolar microtubules slide past each other
What happens during telophase?
Mitotic spindle dissociates, vesicles containing original nuclear membrane assemble around the two chromosomes and lamins are dephosphorylated to form new nuclear membrane
What phosphorylates nuclear lamins?
M-CDK
Where do interpolar and astral microtubules go?
Interpolar goes spindle pole > spindle pole and astral goes spindle pole > cell membrane
What catalyses the condensation of DNA into chromosomes?
Cohesin and condensin
What do cohesin and condensin do?
Cohesin holds the chromatids together, condensin coils then
What does the position of the cleavage furrow depend on?
Astral and interpolar microtubules
Why does the cleavage furrow form?
Contractile ring of actin and myosin which contracts the ring and pinches it off
What directs the assembly of the cleavage furrow?
RhoA
Signalling between what generates the zone of RhoA?
Anaphase spindle and cortex
What does contraction of the central ring form?
Midbody which organises intercellular ridge
What catalyses membrane scission either side of the midbody?
ESCRT-III
How many units in a centriole?
9
When are centrioles duplicated?
Duplicated in G1 phase
How does the spindle assembly checkpoint trigger mitotic exit?
Degrades M-CDK (degrades cyclin B and securin)
What can defective M phase checkpoints cause?
Chromosomal instability and aneuploidy
What is the process of apoptosis?
Cells become rounded and retract from neibouring cells, condensation, hydrolysis and fragmentation of nucleus and DNA, golgi, ER and mitochondria fragment and cytochrome c is released, “eat me” signal displayed, cellular detachement, phagocytosis
What is exposed as an “eat me” signal?
Phosphatidylserine
What controls intrinsic apoptosis?
Apaf and caspase 9
What controls extrinsic apoptosis?
Caspase 8
Which caspases are activated during a normal tissue injury?
Caspase 3/7
What is released during a tissue injury to regenerate tissues?
PGE2
What does Apaf-1 lead to?
Caspase release
What stops production of intrinsic apoptotic factors?
Bcl2
What does the apoptosome do/contain?
Caspase-activating complex containing caspase 9 and Apaf-1
What do initiator caspases ONLY affect?
Executioner caspases, itself and “BCL-2 homology 3-interacting domain death agonist (BID)”
Which caspases are the initiator caspases?
2,8,9,10
How are caspases activated?
Cleavage
Which are the executioner caspases?
3,6,7
Which tetrapeptide motif do caspases cleave substrates after?
X-X-X-Asp
How are initiator caspases activated?
Inactive monomer > dimerisation and interchain cleavage > active
How is activation different in caspase 9?
Cleavage inhibits rather than activates it
How are executioner caspases activated?
Present as dimers > cleaved > intermolecular rearrangements > enzymatically activated dimers
Which caspases aren’t used for apoptosis and what do they do instead?
1,4,5,12 during innate immune responses (regulating inflammatory cytokine processing)
How do initiator and executioner caspases exist in cells?
Monomers, preformed dimers
What subunits do all caspases have?
Propeptide, large and small subunit
What structure do caspases function as?
Heterotetramers
What do pro-apoptotic BCL-2 proteins do to mitochondrial membrane permeability?
Either cause it or just relay the signal
Which domain do all BCL-2 proteins have?
anti-apoptotic, pro-apoptotic effector and pro-apoptotic BH3 effector
Is BCL2 pro-apoptotic or anti-apoptotic?
Both
What regulates Noxa and Puma?
p53
Are bax/bak pro or antiapoptotic? What do they cause?
Pro-apoptotic because they cause cyt c leakage
What cancer types is Bcl-2 expressed in?
Non-hodgkins lymphoma
What do death stimuli induce the formation of?
Bax/bak pore (and release of cyt c, smac and diablo)
What can caspase 8 lead to the direct cleavage of? What else can it do?
Caspases 3/7 OR can indirectly cleave Bid adn then stimulate Bax/Bak
What does XIAP inhibit?
The apoptosome and caspase 3/7
What inhibits XIAP?
smac, DIABLO, HtrA2 and Omi
What does the TNFalpha receptor recruit to form complex I?
TRADD, TRAF2 and RIP1
What does complex I recruit to form complex II?
FADD and caspase 8/10
What follows complex II formation?
Apoptosis
What does FLIP bind to and inhibit?
Caspase 8
What domains do caspases interact with and what stops this?
BIR
How does the apoptosome form?
Start with Apaf-1, cyt c then binds to WD40 domains, CARD domain is released, 7 Apaf-1 monomers form a heptamer, caspase 9 recruited to CARD domain, caspase 3 activated
What domains do cytochrome c bind to?
Wd40
Which capase is recruited to the CARD domain?
9
What is MDM2 and what does it inhibit?
A ubiquitin ligase which inhibits p53
What are the anti-apoptotic Bcl-2 proteins?
BCL-2, BCL-XL, MCL1, BCL2A1, BCLW
How do Bcl-2 proteins block apoptosis?
Prevent oligomerisation of Bak and Bak in the outer membrane so there’s no cyt c efflux
What BCL2 proteins do BID and BIM interact with compares to NOXA and PUMA?
BID/BIM interact with all antiapoptotic, NOXA/PUMA only interact with some
How does p53 affect NOXA/PUMA?
Upregulates them
What does proteolysis of BID by caspase 8 generate?
tBID
How do you treat BCL2 overexpression?
Use BH3 mimetic compounds (natural antagonists of BCL2)
Which BH3-only proteins promote apoptosis when overexpressed?
BID, BAD, BIM, BIK, BMF, NOXA, PUMA, HRK
What is a metacentric chromosome?
Centrosome is in the middle
Which kind of DNA does the dye show?
Packed DNA
Which bases are found in lighter-dyed DNA?
AT
What is a telocentric chromosome?
Centromeres at tip eg mouse
What are Mendel’s first, second and third law?
1 - principle of segregation, 2- principle of independent assortment, 3- principle of dominace
What kind of inheritance do polling and Ehlers-Danlos show?
Autosomal dominant
How do you tell if you have an F1 homozygote or an F1 heterozygote?
If you cross the heterozygote to the recessive you would get a 2:1 ratio
What is variable penetrance?
Not all individuals with the genotype display the phenotype
What is variable expressivity?
Individuals with the genotype vary in the extent to which they express it
How do you tell if two genes are linked?
DO a test cross and then use Chi-squared or RF/distance between genes
How do you get yellow labradors?
Amount of pigment = B (eumelanin), Pigment at all = E (MC1R), E is epistatic to B so yellow if ee regardless of B
What is the recombination requency for independent assortment?
50%
What is one centimorgan?
1% recombination = 1 map unit
How many recombination events per chromosome and how many chromatids does it involve?
Multiple, can involve more than two
What does a linkage map quantify?
The relationship between recombination rate and distance between genes
How do you work out recombination frequency and % recombination?
Frequency = number recombinants / number total, x100 = %
Which points can you use to calculate RF? Which is more accurate?
Can either use the two furthest points of the distance between each of the points, second way is more accurate
How do you know which genes are associated with a disease?
Put markers near known disease-causing gene locations and markers that segregate are tightly linked and associated with the disease
What are the two possible recombinants of a in abc?
a+bc and ab+c+ (same b and c, different a)
Where does exon sequencing pick up mutations from?
Only proton-coding genes, not regulatory regions
How do you sequence an exon?
Chop genomic DNA, use a “Human Exome Array” to capture exons, elute these from the array and use PCR to look for mutations
What mutation do Belgian Blue cattle have?
11bp exonic deletion causing framshift in myostatin which is a negative regulator of muscle growth
What is the difference between polyploidy and aneuploidy?
Polyploidy is multiples of ALL chromosomes, aneuploidy is additional individual chromosomes
What is Turner’s syndrome?
45XO
What is Klinefelter’s syndrome?
47XXY
What is XYY male syndrome?
47XYY
What is Down syndrome?
trisomy 21
What is Edward’s syndrome?
trisomy 18
Which aneuploidies are viable? How can you survive others?
Only small autosomes, can survive larger ones if there’s mosaicism
What does non-disjunction during meiosis I cause?
Uniparental heterodisomy and nullisomy
What does non-disjunction during meiosis II cause?
Uniparental isodisomy and nullisomy and normal gametes
What is triploidy usually caused by?
Dispermy or occasionally a diploid gamete
What does first trimerster screening look for?
Low PAPP-A, high HCG and nuchal fold thickening
What are the usual chromosomal abnormalities in spontaneous abortions?
Numerical eg trisomy
How do you get tetraploidy and what is this called?
Failure to complete first meiotic division even though DNA has replicated (called endomitosis)
What is a derivative?
A chromosome carrying a translocation
What can be a problem in carriers of balanced translocations?
Chromosomal abnormality in offspring eg. translocation in Chr 21 can cause offspring with Down syndrome
What is the most common microdeletion syndrome, what is it caused by and what does it cause?
22q11.2 deletion syndrome from non-allelic homologous recombination, causes schizophrenia, neurodevelopmental abnormalities, cardiac and craniofacial abnormalities
What do you use to stain chromosomes?
G banding
What are Robertsonian translocations and what is their effect?
Breaks at or near centromere in two acrocentric chromosomes, get a single dicentric chromosome and a fragment with no centromere, can’t do meiosis and may get unbalanced gametes (trisomy or monosomy)
What is Philadelphia chromosome and what is it common in?
Reciprocal translocation between chromosomes 9 and 22, common in chronic myeloid leukaemia
What is the process of Philadelphia chromosome?
ABL oncogene on 9q juxtaposed to BCR on 22q, the hybrid gene encodes novel chimaeric protein in leukaemia cells, consistent activity causes neoplastic transformation
What is a chromosomal inversion?
Chromosome breaks in two places and the bit in between flips
What is a pericentric inversion?
Break is on either side of the centromere
What is a paracentric inversion?
Only one arm of the chromosome breaks
What is an X-linked gene in males?
hemizygous
What determines sex in placental mammals?
The Y chromosome
How is Duchenne muscular dystrophy inherited and what does it cause?
X linked recessive, mutation in dystrophin gene which produces a rod-shaped cytosolic protein whihc anchors membrane to scaffold
What is Rett syndrome and how is it inherited?
X linked dominant, no affected males because all die and girls survive because X inactivation. Mutation in MeCP2 (chromatin associated binding protein)
How does Array Comparative Genomic Hybridisation work?
Detects microscopic duplications and deletions, test DNA has green fluorescence and the aray is red so duplications show up green and deletions show up red
What is equine Turner’s-like syndrome?
XO aneuploid mare, sterile, no folliculogenesis because of dosage effect on X
What is Von Willibrand’s disease?
X linked haemophilia in dogs (factor VIII deficiency)
What is haemophilia A?
Factor VIII deficiency
What is haemophilia B?
Factor IX deficiency
What is needed to produce a male?
Testosterone and AMH
What does presence of testes cause and what happens if they are removed?
Male differentiation, female phenotype
How do birds adopt sexual identity?
Sex chromosomes not gonadal hormones
What is an XX male?
Translocation of sex-determining gene on Y
What is an XY female?
Missing sex-determing gene?
What is SRY?
The testes determing factor
What is the Y chromosome like?
Repetetive heterochromatic region, repeats, palindromes, hairpins, gene poor
What are the areas of the Y chromosome shared with the X called?
Pseudautosomal region
What do Sox9 mutations cause?
Campometic Dysplasia with XY sex reversal
Where is the mutation in Campometic Dysplasia?
Chr 17, q24
What does the chromosome to be inactivated express?
Xist (a non-coding RNA
What is ovarian teratoma?
Germ cell tumour of egg, parthenogenetic 46XX (maternal)
What does Sf1 regulate?
SRY during primary sex differentiation
Which complex is involved in the male sex-determining pathway?
Sf1-Sry-Sox9
What is heteropyknotic X?
Inactive, random, stable, inherited
What is parthenogenesis?
Asexual repro
What is a complete hyatidiform mole?
Diploid paternal 46XX
What escapes inactivation at the tips of the sex chromosomes?
PAR1 and PAR2
What is Prader-Willi syndrome?
mUPD15, missing paternally expressed imprinted genes
What is maternal uniparental disomy?
Disomic egg x mullisomic sperm
What is paternal uniparental disomy?
Nullisomic egg x disomic sperm
What is Angelman syndrome?
pUPD15 (maternally expressed imprinted gene UBE3A missing)
What is Beckwith-Wiedemann syndrome?
Paternally inherited chromosome II (pUPD11), altered dosage of IGF2 growth factor, mutated expression of CDKNIC cell cycle repressor
What is Callipyge sheep?
Imprinted disorder, mutant allele must come from father
What methylates DNA at which carbon?
DNA methyltranseferase at C5
What does pericentric chromatin require for normal function?
Repressive histone modifications
What are characteristics of an “on” gene?
Euchromatin, unmethylated, acetylated histones, H2K4 methylations
What are characteristics of an “off” gene?
Heterochromatin, cytosines sometimes methylated, deacentylated histones, H3K9 methylation, H3K27 methylation
Where is maternal methylation in imprinting?
At promoters of imprinted genes
Where is paternal methylation in imprinting?
Between imprinted genes
Do oocytes or sperm have more mitochondria?
Oocytes
What is the difference between homoplasmy and heteroplasmy?
All mitochondria identical vs normal and mutant strains within the cell
What can heteroplasmy cause?
Intrafamilial variation in mitochondrial diseases
What is found in discordant diseases in twins?
Differentially methylates regions (DMRs)
What is mitochondrial DNA like?
2-10 copies of it, no introns or repeats, mostly functional gene products, multigenic transcription
What recruits Dnmt3 to imprints?
Sequence-specific DNA binding protein called ZFPS7
What is depleted int eh pre-implantation embryo?
Dnmt1
What is recruited to imprints int he pre-implantation embryo?
Dnmt3 (de novo)
What is fragile X syndrome?
X linked trinucleotide repeat in FMR1 gene causing overmethylation of DNA, all daughters of transmitting male are carriers
What can you use to mutate or correct genes?
CRISPR-Cas9
What is cas9?
An RNA-guided dsDNA endonuclease
What do trinucleotide repeast at full mutation range attract?
Epigenetic silencing
What are the three types of trinucleotide expansion diseases?
Loss of function due to block of transcription, gain of function by pathogenic RNA or gain of function by a mutant protein
What is Friedreich’s ataxia
Autosomal recessive on chr 9, GAA expansion repeat in intron 1 of Frataxin gene which recruits repressive marks and reduced transcription, NOT methylation-mediated silencing
How do you treat Friedreich’s ataxia?
Reactivate frataxin using histone deacetylase inhibitor
What is myotonic dystrophy?
Autosomal dominant with anticipation, unstable CTG repeat in 3’ end of MD protein kinase, doesn’t affect protein coding and isn’t in 5’ regulatory region, acts as pathogenic RNAso toxic gain of function of RNA binding proteins
What can polyglutamine aggregates for and what can this cause?
Antiparallel/parallel noodles, B sheets, cyclinders or hairpins which can damage mitochondria, induce ROS, induce apoptosis, Ca2+ may activate caspases, positive feedback loop
What is Huntington’s disease?
CAG (glutamine), chr 4, aggregates in neurone, more unstable in males
What is genetic drift?
Alleles/variants being lost from the popn at random
What happens once prevalence of an allele reaches 100%
No more change
When is heterozygosity highest with respect to p and q?
When p = q = 0.5
What is Ellis Van-Creveld Syndrome?
Single gene disorder from founder effect
What is a “selective sweep”?
As positively selected alleles rise in frequency, linked alleles on the chromosome move and create a selective sweep
What is purifying/negative selection and where is it strongest?
The removal of deleterious alleles, strongest on protein-coding sequences, moderate on flanking sequences and weak on intergenic elements
What is balancing selection?
Where multiple alleles are actively maintained at frequencies above that of gene mutation (recessive gene could have advantage)
What is a non-synonymous substitution?
A non-silent mutation
What is synonymous substitution?
A silent mutation
What is single nucleotide polymorphism?
Any single site in the genome where two or more different nucleotides are segregating in a population
What is a haplotype?
A distinct combination of defined alleles/variants/SNPs inherited together
What does the HapMap project do?
Identifies haplotype variants in populations to assess correlation between genotype and phenotype
What do long terminal repeats do?
Mediate integration into the host cell genome (contains gag-pol-env)
What does gag do?
Group-specific antigens (viral capsid proteins)
What does pol do?
Reverse transcriptase and DNA integrase
What does env do?
Viral envelope proteins
What do DNA integrase do and what does it need?
Looks for LTR and insertes dsDNA there, needs a strong promoter region so mRNA is produced
What is v-src and what is c-src?
c = cellular, v = viral (hijacked mutant version which is always active)
What does SH3 bind?
Polyproline
What does SH2 bind?
Phosphorylated tyrosine
What is the structure of inactive src?
C-terminal Tyr527 bound by internal SH2 domain
What is the structure of active src?
Binding of G protein or dephosphorylation of of Tyr527 displaces SH2
What does src do?
Kinase which activates ras
What has the rous sarcoma virus got?
Intact viral genome And oncogenic src
What happens to src at high temperatures?
No functional, cells revert to normal phenotypes
Which critical Ras residues are associated with GTP binding?
12, 13, 61
What happens in non-AIDS Burkitt’s lymphoma?
c-myc is deregulated by chromosomal translocation (oncogenic myc steals strong promoter region of antibodies)
What do retroviruses do to the c-myc gene?
Insert an LTR promoter at each end
What happens if the virus takes all the src gene?
Properly regulated using Tyr527
What happens if the virus doesn’t take all the src gene?
Doesn’t have a tail, always activates Ras and always causes proliferation (deletion of carboxy terminus)
How does MDM2 inhibit p53?
Covalent modification
What inhibits MDM2?
p19
What is myc expressed in response to?
Growth factors
What does myc do?
Transcription factor for genes involved in proliferation
WHat does myc do to Bcl-2?
Inhibits
How can myc be deregulated?
Genomic amplification (DNA pol slipping), chromosomal translocation of retorviral insertion
What do heterozygous Rb alleles show?
High frequency of loss of heterozygosity
What does BRG1 do?
Keep histone in position on the gene?
What does Bad inhibit?
Bcl-2
How does Ras cause cell cycle arrest?
Upregulates p16
What phosphorylates Rb so E2Fs aren’t sequestered?
CDK4/6
What is a multicopy gene?
A gene with multiple copies in the genome eg snRNPs
What happens in Frasier syndrome?
SOme WT1 proteins have KTS, some don’t - the ratio is 2:1 in normal cells but 1:2 in Frasier syndrome. It’s a transcription factor and the balance of the two forms is needed for function.
How is siRNA formed?
Long dsRNA bind to dicer and is cleave to produce small dsRNA fragments (the siRNA)
What is the RISC complex formed from and what is the catalytic part?
Dicer, argonaute 2 and TRBP (helps to bind RNA). Argonaute 2 is the RNase catalyst.
What does RISC do?
Degrades and destroys mRNA so protein expression is decreased
What happens if G1/S arrest?
p16INK4A to prevent CDK4/6 from binding cyclin D
What phases do licensing and firing occur in?
Licensing G1, firing S
How does negative T cell selection occur?
Medullary thymic epithelial cells (mTECs) display “self” Ags to developing T cells, this signals self-reacting T cells to apoptose and are deleted from repertoire.
What are T cells that aren’t self-reactive called?
Self-tolerant
How are self-tolerant T cells activated?
Must encounter self cells with surface protein complex called MHC that is presenting an antigen
What does everything in the BH family have?
At least one BH domain
What are the four types of BH domain?
BH1,2,3 and 4
How many BH domains do BCL-2, BCL-XL, MCL1, BCL2A1 and BCL-W have?
Four
What does BCL-XL do?
Binds to Bax/Bak and causes cyt c release
How does DISC form?
CD95L binds to CD95, FADD recruited, activates pro-caspase 8, inhibit apoptosis
How do decoy receptors inhibit DISC?
Resemble CD95 but don’t actually do anything
How do FLIPs inhibit DISC?
Inhibit interaction between CD95, FADD and pro-caspase 8.
How do IAPs inhibit DISC?
Bind and inhibit caspases activated by it
What is copy number variation?
Where stretches of the genome are duplicated etc. in some individuals but not in other
What are low copy repeats?
Repetetive DNA at several points in the genome
How do you do FISH chromosome painting?
Design probes complementary to the DNA you’re interested in. Incubate it with the DNA and then look for fluorescence to see if that sequence is in your sample.
What are some ways to repair wild type p53 function?
Use adenovirus to infect tumour cells and force them to express it, use drugs to change the mutant p53 shape back to the right one, adenovirus lacking EIBp55 can’t replicate in cells containing normal p53 so if it infects a tumour it will only kill cells with mutated p53
How can you stabilise the p53 pathway?
Small molecule called RITA binds to p53 and blocks p53-MDM2 interaction, so MDM2 can’t bind and target eh p53 for degradation. Nutlins do this too
What part of the DNA isn’t spliced properly in Frasier syndrome?
Intron 9 is psliced and removed from the mRNA and extra mRNA from the exon is lost which codes for lysine-threonine-serine
What phosphorylates pRB?
Cyclin D - CDK4/6 and later Cyclin E - CDK2, releases E2F TF and allows transcription of S phase proteins
What is proteolysed at the start of S phase?
Cyclin D
