Molecular And Cell Biology Flashcards
What is Ferritin?
A protein which stores, transports and releases iron.
What is Porin?
A protein which sits in the outer bacterial membrane - allows diffusion of certain molecules.
What is a protein’s secondary structure?
Has hydrogen bonding.
Alpha helix - h bonds between amino and carboxyl groups (4 residues apart)
Beta sheet - h bonds between different strands.
What is a protein’s tertiary structure?
Thermodynamically stable - 3D.
Determined by non-covalent interactions.
Where do amino acids go in a tertiary structure?
Polar residues end up on the outside - so can interact with polar water molecules.
Non - polar fold in centre.
What is a disulphide bridge?
Interaction between sulphur atoms in cysteine.
What is a protein domain?
Some proteins fold into different domains - separated by flexible regions.
They carry out a specific function.
What is a protein’s quaternary structure?
Formed of subunits.
2 = dimer, 3 = trimer, 4 = tetramer.
What is methylation?
Post-translational modification
Adds on a -CH3 group.
E.G. on histones to control genome expression.
What is glycosylation?
Post-translational modification
Adds on sugars.
What is ubiquitination?
Post-translational modification.
Adds a 76aa polypeptide to mark protein for degradation.
What is phosphorylation?
Reversible
Adds PO3 - uses kinase enzymes.
Regulates enzyme function.
What is protein targeting?
Some proteins contain signals or localisation sequences to show where they need to go.
Some are targeted to cell membrane by the secretory pathway (become channel proteins ect).
What are anchor membrane proteins?
Anchored to membrane by additional hydrophobic groups added on - allows them to be be removed from membrane.
E.g. Ras
What is a microtubule?
Made of alpha and beta tubulin
Is there rotation around peptide bonds?
NO
What is a short chain of amino acids called?
Peptide
What is an unfolded protein called?
Denatured then turns native (folded)
What are the Mendelian laws of inheritance?
Segregation: genes come in pairs, one is passed on to offspring.
Independent assortment: genes are passed on separately from eachother.
Dominance: the dominant allele will be expressed.
What was Sutton and Boveri’s chromosome theory of inheritance?
Observed meiosis in grasshoppers and worms (as chromosomes are large and few) - noticed that destroying chromosomes stopped normal embryo development, consistent with mendel’s law
- Chromosomes are required for embryonic development.
- Chromosomes carry ‘Mendel’s factors.
- Chromosomes are linear structures with genes along them.
What is streptococcus pneumoniae?
Causes pneumonia in humans and mice - only some strains.
S strain - smooth, polysaccharide coats - pathogenic.
R strain - rough - no coat - not pathogenic.
The coat forms a capsule which protects strains.
What did Griffith do to the streptococcus?
Heated S strain - no infection
Heated S strain and R strain - infection
This due to R cells transforming as some material from s –> r.
This is the transforming principle.
What did Avery, Macleod and McCarthy do?
No one knows what was passed to R cells.
They destroyed different parts to see what was causing it.
They found it was small pieces of DNA which coded for the capsule.
What is a bacteriaphage?
A category of viruses which require bacterium to be a host cell.
What is bacteriophage T2?
The host is Escherichia coli.
It destroys the chromosomes and replicates its genone.
What experiment did Hershey & Chase do which showed how bacteriophages reproduce?
1) label bacteriophage DNA or protein with radioactive isotope
2) Infect a bacteria (only DNA enters)
3) Separate phage ghosts and bacteria (use blender and centrifuge)
4) test for radioactivity
How did they label bacteriophage with radioactivity?
32P and 35S are unstable isotopes - can be detected by Geiger counter.
Growing the bacteriophage in media with them will produce radioactively labelled protein/DNA.
What did the bacteriosome experiment show?
Supernatant had phage ghosts, pellet had bacteria. They were tested for radioactivity - It showed that DNA was injected, not proteins.
What are the purines and pyrimidines?
Purines - A,G (2 carbon rings)
Pyrimidines - C,T,U (1 carbon rings)
What is a nucleoside?
Sugar + base
What did Chargaff do?
Used chromatography to seperate and isolate nucleobases.
Chargaff’s rules
%A = %T, %C = %G
%AT does not = %GC
What are the main features of Crick and Watson’s model?
A-T and G-C hydrogen bonded pairs
Antiparallel
Right handed double helix
Major (big gap) and minor grooves (little gap)
One helical turn every 10.5 bp
How many hydrogen bonds between AT and CG?
AT = 2
CG = 3
5’ to 3’ polarity
What maintains the DNA width?
The binding of one purine and one pyrimidine
What is one complete turn of DNA?
3.4 nm, 10.5 bp
What is a centromere?
Specialised region where microtubules attach - doesn’t have to be in the centre
What is a telomere?
Repetitive DNA at end of chromosomes
Protect the ends of chromosomes
What is the prokaryotic genome?
Single, circle chromosome
Plasmids also found
Passed between cells by conjugation
Few million bp
What are DNA-binding proteins?
Have domains which can regulate gene expression, cut DNA and protect DNA
e.g. restriction endonucleases, transcription regulators, histones
What is a transcriptional regulator?
Proteins which bind to reg sequences near promotors to stimulate or block transcription. Bend DNA in favourable or unfavourable ways
e.g. lac operon - lac repressor binds to DNA to block transcription
What is a restriction endonuclease?
Enzyme which cuts DNA at specific sequences.
Restricts action of viruses.
Bacteria is protected as methylated
What are histones?
Proteins which chromatin is wrapped around.
What are the bases and nucleosides in RNA?
Uracil - uridine
Adenine - adenosine
Guanine - guanosine
Cytosine - cytidine
What are stem-loop structure?
Short helices in RNA caused by intramolecular base-pairing.
Secondary structural elements.
e.g. tRNA
What do most interactions in RNA occur in?
Minor groove - major too narrow
What is non-canonical base-pairing?
G-U, A-C - wobble base pair
These can stabilise RNA
Why is deoxyribose more stable than ribose?
Lacks a OH on second carbon
The OH makes ribose more reactive and prone to hydrolysis
What is the secondary and tertiary structure of RNA?
Secondary - 2D map defined through intramolecular base-pairing.
Tertiary - interactions that connect regions separated in secondary structure - can be canonical.
What is the A minor motif?
Two adjacent A bases interacting with the edge of a G-C base pair.
How is RNA made?
From RNA polymerases - transcription.
RNAP active site contains a short RNA/DNA heteroduplex
Where does RNA polymerase start?
Promotor regions until reaches terminator region.
What is the E.coli RNA polymerase core enzyme?
Protein complex containing 5 subunits:
2 alpha - binds transcription factors
2 beta - catalytic
w - assembly and stability
What are sigma factors?
Provide specificity to RNAP for the gene promotor.
In prokaryotes
Released from RNAP
How many RNA polymerases in eukaryotic cells?
I = rRNA
II = mRNA, noncoding RNA
III = tRNA, 5s RNA
Have conserved core structure
What is a gTF?
General transcription factor
Required to assemble RNAP II onto promotors in eukaryotic cells.
A preinitiation complex involves a multi-step pathway.
e.g. TFIIA, TFIIB
How did Wilkins and Franklin see DNA?
X-ray crystallography - saw x pattern (helix), regular pattern, distance between spots (one turn) = 3.5 nm
What did Meselson and Stahl do?
Used nitrogen isotopes to experiment semi-conservative DNA.
14 and 15 - nitrogen
1) grew the bacteria in media to make heavy (15) and light (14) DNA
2) separate by ultracentrifugation
3) mix with caesium chloride
4) look at DNA using UV light
5) saw different bands after every generation
1st - 1 band
2nd - 2 bands
3/4/5 - 2 bands but hybrid getting lighter and lighter
How many replication origins?
1 in E.coli
Tens of thousand in humans - bidirectional replication forks
What does DNA polymerase do?
Adds nucleotides one at a time 5’-3’ using template strand
What does primase and ligase do in DNA replication?
Primase generates the primer
Ligase joins the new DNA together (loose ends into a single strand)
What do topoisomerase and helicase do in DNA replication?
Topoisomerase relieves pressure from overwinding by breaking and resealing DNA.
Helicase breaks H bonds between two strands
What is the single-strand binding protein?
Prevents the two DNA strands from reannealing in DNA replication
What are the leading and lagging strands?
Leading strand - DNA points towards replication fork - continuous
Lagging - DNA points away so must be discontinued and primed multiple times
BOTH 5’-3’
What are okazaki fragments?
Pieces of DNA - they are stuck together to make lagging strand
Why is there erosion at the end of chromosomes?
Primer is removed - leaves a gap so lagging strand is incomplete - telomeres solve this - telomerase can replenish telomeres
How are RNAs resolved?
Large (rRNA) - in agarose gels
Small - acrylamide gels
mRNA is not clearly seen
What are the major cellular RNAs?
mRNA - 5%
rRNA - 75%
tRNA - 10%
How is mRNA processed?
- capped at 5’ end
- pre-mRNA splicing
- 3’ end processed (cleavage and polyadenylation - adding a stretch of adenine nucleotides)
- happens in nucleus
- the cap and poly(A) protects and promotes translation
What is the m7G cap?
A guanosine nucleotide is added to the 5’ end of RNA pol II transcripts - linked by 5’5’ triphosphate linkage.
Then methylated
What is polycistronic?
Where an mRNA can code for multiple proteins - in prokaryotic cells
What are split genes?
Genes are interrupted by introns
Prokaryotic cells DO NOT HAVE THIS
How is pre-mRNA splicing accurate?
Introns and exons have splice site sequences
5’ - GU (start of intron)
3’ AG (end of intron)
Introns also have a branchpoint ‘A’
What causes splicing?
Carried out by a ribonucleoprotein (RNP) called spliceosome
These are made from smaller RNA/proteins called snurps
What are the steps in splicing?
Involves 2 transesterification steps -
1) 5’ exon removed and the intron forms a lariat structure involving branchpoint adenosine
2) exons joined and intron lariat released
Can some RNA’s undergo self splicing?
Yes!
Nuclear splicing is said to have evolved from this
Enzymes with RNA catalytic subunit - ribozyme
What does understanding the genetic code allow us to do?
- infer protein sequences from DNA and its sequence
- infer protein function by comparison
- design tools to study protein function e.g. drugs
How are mRNA codons recognized?
By base pairing with anticodons within cognate tRNAs - the tRNAs are charged/aminoacylated (connected to appropriate amino acid) - mediated by aminoacyl-tRNA synthetases
How does the amino acid on tRNA bind to the chain?
Peptidyl group and aminoacyl groups bind - peptide bond
What’s the name for ‘all nucleotides have a genetic meaning’?
Nonpunctuated
Some have same meaning - degenerate
How was the genetic code cracked?
Cell extracts were programmed to make proteins using artificial RNAs made with polynucleotide phosphorylase.
e.g. poly(u) RNA made phenylalanine
How many codons does the genetic code have?
64 - 61 are sense = code for amino acids
- 3 are stop codons - UAA, UAG, UGA
The start codon is AUG - methionine
Met and Trp have unique codons - others degenerate
What are synonymous codons?
Code for same amino acid - usually differ by 3rd nucleotide
Some recognised by isoacceptor tRNA’s - charged with same amino acids
Others can be recognised by the same tRNA - wobble base-pairing
What nucleotide do many tRNA’s have?
Inosine - 1st position on anticodon
Can pair with U,A,C
G can pair with C or U
What is the tRNAs secondary structure?
Cloverleaf - 5’ and 3’ drawn together
Amino acid is bound to 3’ hydroxyl on A nucleotide
All tRNA have 3’ terminal CCA
Modification of 1st position of anticodon allows wobble
What is coaxial stacking?
Increases thermodynamic ability in tRNA
Anticodon on D arm, acceptor on TYC arm
Base pairing between the arms
How is the tRNA charged?
Mediated by aminoacyl-tRNA synthetases - needs ATP
20 different ones - causes ester link between carboxylic group of AA and 3’ hydroxyl group
What is the ribosome structure?
Large RNP particle - has two unequal subunits
Codon/anticodon occus on small subunit
Peptide bonding occurs on large subunit
There are 3 tRNA binding sites (A,P,E)
Has a peptidyltransferase centre (PTC) - RNA rich - makes peptide bonds - RNA catalysed
How are ribosomes synthesised?
rRNA transcription and early steps in nucleoli
Later steps in nucleosomes and cytoplasm
What happens during translation elongation?
2 tRNAs are bound at a given time, either at A & P, or P & E.
Contains cycles of:
- aminoacyl-tRNA binding
- peptide bond formation
- translocation of the ribosome along mRNA
When aminoacyl-tRNA binds to A - tRNA is released from E
What do A, P and E mean in ribosomes?
P = peptidyl site
A = aminoacyl site
E = exit site
How are GTPases used in translation elongation?
The aminoacyl-tRNA brought to ribosome by the elongation factor EF1A (EF-Tu in prokaryotes)
Translocation needs EF2 (EFG in prokaryotes)
Both are GTPases - 2 GTP molecules are hydrolysed per cycle
How does the tRNA go to the start codon?
EF1a brings Methionyl-tRNAs to bind to AUG codons and associate with eukaryotic initiation factor 2, eIF2 (IF2 in prokaryotes)
What is the Shine-Delgarno sequence?
Allows the start codon to start at P site - in prokaryotes - near the start codon - 3’ end of 16s rRNA
How is translation initiated in eukaryotes?
tRNAmet (bound to eIF2) and 5’ mRNA bind through interaction with cap-binding complex.
The preinitiation complex scans along mRNA - finds AUG - kozak sequence
The large subunit is then recruited
How is translation terminated?
Eukaryotic release factor 1 (eRF1) or RF1/2 in prokaryotes trigger peptide hydrolysis .
eRF3 (GTPase) releases peptide by releasing RF1/2 from ribosome
What are housekeeping genes?
Genes that are constitutively expressed
What mutations can effect regulatory elements?
cis - mutation within the same gene - DNA sequences that effect gene regulation
trans - in a different gene - protein or RNA factors that regulate the expression of target gene
What stage are genes mostly regulated?
Transcriptional
What are transcription factors?
Can activate (activators) - positive control - at weak promotors
Can repress (repressors) - negative control
Activators in E.coli interact with a subunit of RNA polymerase - promote binding
How are transcription factors modulated?
Inducers - bind to TF and stimulate activators or inhibit repressors
Corepressors - bind to TF and stimulate repressors or inhibit activators
What is the lac operon?
Codes for 3 genes - lacZ, lacY and lacA
Controlled by transcription repressor gene - lacI
LacI - binds to operator (lacO) and blocks RNA polymerase
Lactose is the inducer - inhibit repressor
Negative - inducible
What is CAP in lac operon?
lac operon also regulated by activator - catabolite activator protein
Binds to lac promotor when associated with cAMP (inducer) - stims transcription
cAMP is inhibited by glucose (ideal energy source)
Lac operon - expressed by absence of glucose and presence of lactose
How can RNA processing control gene expression?
Pre-mRNA can occur in different patterns - different proteins
How does translation control gene expression?
Translation is downregulated by integrated stress response
Can also be regulated by specific mechanisms e.g. Ferritin expression is responsive to Fe2+
How does post-translational modifications regulate protein function?
Most commonly - phosphorylation of serine, threonine or tyrosine by kinases
How is translation inhibited by eIF2a?
eIF2 hydrolyses GTP to GDP - regeneration requires eIF2B but eIF2a is phosphorylated by integrated stress response and binds very tightly to it so it cannot be recycled
What are the basic steps of molecular cloning?
1) insert DNA into a vector (usually a plasmid) - makes recombinant DNA
2) transfer to host
3) replicate
What features must a vector have?
1) origin of replication - replication in host
2) selectable marker - survival of host cells e.g. antibiotic resistant gene
3) multiple cloning site - where we insert gene - has restriction enzyme sites
How do we cut up DNA?
Use restriction enzymes - recognise short specific sequence
There are 3/4 types:
- types I and III cleave DNA at random places far from recognition sequence
- type IV cleave modified DNA
- type II cut DNA at a specific place
Name them: Species, Strain, Enzyme e.g. EcoRV
What are type II restriction enzymes?
Most commonly protein homodimers
DNA sequence usually palindromic
Recognise specific DNA sequence
Can generate overhangs or blunt edges - overhangs are compatible
How do type II restriction enzymes work?
1) initial binding is non-specific
2) moves along until finds specific site
3) this binding causes structural changes
4) catalysis requires Mg2+
5) generates 5’ phosphate and 3’ OH ends
What is the ligation reaction?
Sticky ends have to interact - DNA ligase catalyses new phosphodiester bond
What problems may molecular cloning have?
- insert doesn’t join vector
- little DNA
- DNA may be mixed into lots of other molecules
How do we modify the DNA ends in molecular cloning?
Removal/addition of 5’ phosphate - need to form phosphodiester so needs to be there
Adding - T4 polynucleotide kinase
Removing - Calf Intestinal Phosphatase (CIP) - this can prevent self-ligation
How can we make blunt ends?
Fill in 5’ overhang or remove 3’ overhang - T4 DNA polymerase or DNA polymerase I
Remove 5’ overhang - Mung bean nuclease
How do we make host cell take in recombinant DNA?
1) electroporation = brief high voltage
2) chemical trasformation - heat shock - causes membrane changes that allow DNA uptake
- not that efficient - use antibiotics to find cells with the DNA
What is PCR?
DNA replication in a tube - DNA doubles each cycle
can solve cloning problems - not having enough DNA and DNA might be mixed with other DNA
What are the stages of PCR?
1) denaturing - DNA dissociates into single strands at high temperatures (95)
2) primer annealing - primers bind to complementary sequence (55-65)
3) primer extension - DNA polymerase synthesises new strands from 3’ end of primer (68-72)
Which DNA polymerase is used in PCR?
Taq - thermostable and has high processivity and extension rate (2-4 kb per minute) however - low accuracy and adds adenine overhang - no proofreading
Pfu - better thermostability, slower extension rate (1kb per minutew), products are blunt ended, more accurate - proofreads
What features do PCR primers have?
- has to be minimum 17 base pairs
- be specific to template
- come in pairs
- appropriate melting temp
What do we need for PCR?
Template
DNA polymerase
Primers
Deoxyribonucleoside triphosphate (dNTPs)
Buffer
Thermocycler
What is the melting temperature (Tm) in PCR?
Temperature that primer dissociates from DNA - 60/64 degrees
- determines what the annealing temp should be (5 less than Tm)
To work out: add 4 degrees for G/C or 2 for A/T
If too low - primers may bind to other parts of sequence
If too high - may not bind at all
Can we put a PCR product into a vector?
Yes! - there’s no phosphate at the end - add T4 PNK, taq has overhang which needs to be removed - T4 DNA pol
We can add sequences at 5’ end which is complementary to restriction enzymes.
What is reverse transcription PCR?
RNA is reverse transcribed into DNA (cDNA - copy/complementary) - this is then amplified with PCR
How do we make cDNA?
reverse transcriptase synthesises the first strand of cDNA:
1) poly(dT) primers bind poly(A) tail of mRNA
2) mRNA removed
This makes first strand
1) synthesised by klenow fragment of DNA polymerase I
2) hairpin formed by RT acts as primer
3) the ssDNA loop can be digested by nuclease
This makes the second
Then do normal PCR
What is quantitative PCR?
PCR but we can see how much DNA at any time
PCR is exponential at the beginning - until things run out
We can measure product by:
- fluorescent dye - SYBR green fluoresces when binds to dsDNA - they are proportional
- fluorescent probe - sequence specific - is bound to DNA until replaced by strand - then fluoresence - we can use several
What is Ct?
Cycle threshold - where fluorescence exceeds background levels
Difference in Ct = relative measure of what sample had most sample. we can do 2^-difference to find difference
Lower CT = more template at start
We can also minus test from sample for A , then B then minus A and B then do 2^-this number
How do we remove our DNA from bacteria?
Mini prep: Grow lots of bacteria - then break them open - plasmids are small - we will have purified recombinant DNA
How do we analyse recombinant DNA?
We can use restriction enzymes to digest the DNA and see if we get the correct sized fragments
We can also use PCR - see if we get the correct product
We can use electrophoresis to separate fragments - add dye (midori green/ethidium bromide)
How can we use restriction sites to analyse recombinant DNA?
If we add restriction sites to the gene and vector, but have different enzymes that will effect them, we can see different patterns if the DNA is not present/inserted wrong
- restriction fragment length polymorphism - been mutated or gotten longer due to disease - won’t bind
How can we use PCR to analyse recombinant DNA?
Use 2 primers: 1 for DNA, 1 for vector
If we get PCR products - the DNA and vector are correctly combined, no product - no DNA or incorrect DNA
- allows us to identify diseases, cancer ect. & genetic fingerprinting
How does Sanger sequencing work?
Allows us to see that there’s no PCR mutations
1) DNA is amplified using one primer
2) ddNTP (dideoxynucleoside triphosphate) is added - stops sequencing as there’s a missing OH (there’s also normal dNTP)
3) If we add one type e.g. ddCTP then we know that’s the last nucleoside
4) we can then use electrophoresis to see how big the fragments are
5) repeat with ddATP, ddGTP and ddTTP
What is modern sequencing?
One reaction has all ddNTP - all have different fluorescent labels - use capillary gel electrophoresis which is read by computer
What are the components of modern sequencing?
- A taq DNA polymerase
- dNTPs
- ddNTPs
- Buffer
- Template DNA
- Primer
What methods can we use to analyse RNA expression?
RT-PCR - we can use different isoforms of mRNA and look at the different types.
Hybridisation-based technique - can use RNA to make a probe (DNA and RNA together)
To see RNA expression we can use northern blots and microarray
To see localisation use fluorescence in situ hybridisation
When we read DNA, which way do we read?
5’ to 3’ so if something was complimentary - make sure we reverse
What is northern blot?
1) RNA is separated by electrophoresis (by size)
2) transfer RNA to membrane to allow for blotting
3) add a probe (radioactive) then wash membrane - unprobed will wash away
This is good for comparing mutated genes ect. - can only use one molecule at a time
What is a microarray?
Oligonucleotides are attached to a spot on a chip - each oligonucleotide corresponds to different gene - we add cDNA (made from RNA) which is fluorescent - we can see where it binds
more mRNA = more fluorescence
They give relative levels e.g. equal cDNA = combination of colours
What is fluorescent in situ hybridisation?
Same as northern blot - but probe is fluorescent and visualised by microscopy - we can see these within cells
RNA localisation
What are reporter genes?
Easy to visualise (fluorescent) or assay (enzyme)
e.g. enzymatic reporter genes: luciferase and beta-galactosidase
- we can use these to see how promotor is regulated and organised - we put it next to the promotor and see how much it is expressed in different conditions
increased expression = increased promoter activity
How do we detect proteins?
Usually by primary antibodies - we also use secondary antibodies (usually have a conjugate) as they recognise the primary antibody
e.g. primary = rabbit, secondary = antirabbit
What is western blotting?
1) proteins separated by electrophoresis
2) transfer to membrane
3) add antibody - binds to specific protein also add secondary antibody which usually has a conjugate which produces light
What is immunofluorescence?
Add primary antibody to cell - and a secondary which has a conjugate which makes light - can see different molecules
What are fluorescent fusion proteins?
Binds to target protein and allows us to track and see them.
Green fluorescent protein was the first
How to see live imaging of protein localisation?
Use cloning - fuse coding sequence of the protein to a fluorescent protein (GFP) - it will be transcribed and we can track the protein.
How can we analyse molecular interactions?
To see protein-protein:
- pull down assay
- immunoprecipitation
- yeast two-hybrid
Protein-DNA
- chromatin immunoprecipitation
What is a pull down assay?
We analyse protein interaction in vitro
Uses fusion proteins which have an affinity to a certain ligand, GST is commonly used which binds to glutathione
1) make recombinant DNA (made of protein and the other protein e.g. GST) by placing in e.coli
2) make cell lysate - break them open
3) bind GST to affinity ligand on a bead
4) wash away any unwanted stuff
5) We then have a purified sample then we can identify what can bind to protein X
What is immunoprecipitation?
Identical to pull down assay but uses antibodies of beads
What is a yeast two-hybrid?
Uses fusion proteins
Transcription factors have separate domains for different functions
DNA binding domain is fused to bait, transcription activation is fused to prey
DNA BD binds to promotor
If reporter gene is transcribed = bait and prey interact
What is chromatin immunoprecipitation?
Used to see protein interacting with DNA
Use antibody to purify protein - assay DNA associated with protein
1) crosslink DNA to protein
2) chromatin fragmentation
3) immunoprecipitation
4) DNA purification - get rid of DNA
5) analyse DNA
What gene causes cystic fibrosis?
CFTR
What is a raft?
cholesterol and sphingolipids form microdomains called rafts - slightly thicker
What are the four major phospholipids?
Phosphatidyl - ethanolamine
- serine (negative)
- choline
Sphingo-myelin
What do optical tweezers allow us to do?
Show us the tensile strength of membranes
What is hypo/hyper/isotonic?
Hyper - higher solute outside - cell shrinks
Hypo - lower solute outside - cell bursts
Isotonic - same
Is the cell membrane symmetrical?
NO! - lipids composition is always different in each half
Why is membrane asymmetricality important?
Blood groups - ABO
Blood group is determined by structure of oligosaccharides attached to sphingomyelin
Terminal sugar of oligosaccharides determines groups
Also needed for coagulation (clot) - phosphatidylserine
Needed for cell recognition - macrophage can identify phosphatidylserine or phosphatidylethanolamine
What terminal sugars are for each blood group?
End of oligosaccharides
O - none
A - GalNAc = N-acetylgalactosamine
B - Galactose
What is an electrochemical gradient?
Established by ionic conc
Combination of membrane potential and conc gradient
What channel mutation causes congenital insensitivity of pain?
Voltage gates sodium channel - SCN9A
What channels are involved in glucose uptake?
Glucose/sodium symporter at apical
Sodium/potassium pump at basal
Glucose carrier at basal
Where did the nucleus come from?
Hypothesis 1 - membrane formed around DNA
Hypothesis2 - endosymbiosis (entered a eukaryotic cell)
How is DNA packaged?
Wrapped around histones - packaged in chromosomes
Before cell division - called chromatin
What do we see when we stain DNA?
In electron microscope:
Heterochromatin - dense staining interphase DNA
Euchromatin (genes more oftenly transcribed) - less dense staining interphase DNA
Nucleolus - highly dense stained RNA
We can also see chromosomes using FISH
Is the nucleus compartmentalised?
Yes!
Chromosomes occupy specific territories within the nucleus - can be identified by chromosomal painting
Is there sub-nuclear organelles?
Yes! - they can move around in an ATP-dependant manner
Nucleolus
Speckles - pre-mRNA processing
Cajal bodies - splicing
PML bodies - storage
What is the nucleolus?
Not membrane bound
- processes rRNA to produce ribsosomes
- collection of rRNA genes, precursor rRNA, mature rRNA, rRNA processing enzymes, snoRNP’s
- also processes other types of RNA - tRNA & mRNA
What is the membrane structure of a nucleus?
Double membrane with pores - supported by a meshwork called the lamina
The lamina ensures that the membrane is asymmetric - may also play a role in gene regulation
What are laminopathies?
Genetic disorders of the lamina in nucleus
e.g. Hutchinson-Gilford progeria syndrome
What are nuclear pores?
Seen by SEM
- controls what enters in a size-dependant manner - particles with mw>50000 cannot enter by diffusion but can by active signal-dependant transport
- signal for entry is by a specific peptide sequence
- can open to 26nm in diameter
What is scramblase?
Equilibrates lipids
It catalyses the flipping of phospholipids
What is flippase?
Ensures membrane asymmetry
Facilitates flipping to cytoplasmic monolayer
What is the asymmetry like in RBC membrane?
Negative charged phospholipids (phosphatidylserine) mainly in cytosolic leaflet
How does phosphatidylserine transfer from extracellullar leaflet to cytosolic leaflet?
Translocase transfers to inner leaflet
Scramblase abolishes asymmetry
Equilibrium favours translocase
What is the ER?
Endoplasmic reticulum (SER and RER)
Form hollow tubes and flatterned sacs - chambers are called cisternae
What are the functions of the ER?
1) Quality control
2) Synthesis
3) Storage
4) Detoxification
What does the SER do?
Phospholipid and cholesterol synthesis
Steroid hormone synthesis
Synthesis of storage of glycogen
Calcium store
What is calcium signalling in acinar cells?
Zymogen granules have enzymes important for digestion
Stimulation -> Ca release -> Vesicle fusion -> enzyme release
How do things get transported between ER and Golgi?
In the form of vesicles and tubules
Vesicles bud off ER and are received by Golgi
The vesicles are coated to help formation, either:
- clathrin
- COPI
- COPII
Coating needs to be discarded before it can fuse with the membrane
How do vesicles reach target?
Through SNARE
Two types:
- v-SNAREs: found in vesicle membrane
- t-SNARE: found in membrane of target
In nerves: SNARE binds to a helical bundle with 3 components
What is the golgi’s functions?
- modifying and packaging proteins
- renewing plasma membrane
- delivery of material to other organelles
Where do vesicles bind and leave golgi?
Bind at cis face - leave at trans face
What is the trans-golgi network?
From golgi to membrane - secretory pathway
From golgi to lysosomes
From golgi to secretory vesicles (storage)
What things can be taken up by endocytosis?
Nutrients
Antibodies
Enzymes
Signals
Viruses
Bacteria
What happens to endocytosed material?
Can be:
- recycled
- degraded
- transcytosis
Membrane is recycled
What are the different endocytic pathways?
Small scale e.g. clathrin
Macropinocytosis
Phagocytosis
What is phagocytosis?
Uptake of large particles - bacteria/apoptotic cells
Pathogens usually coated by antibodies - opsonization
Can be frustrated - when target is too large and two macrophages are trying to engulf
- ligand coated particle binds to phagocyte surface receptor, cell extends pseudopods to engulf particle
What is macropinocytosis?
Cells form actin driven ruffles which can fuse to form macropinosomes
Similar to phagocytosis
Not selective
Used by cancer cells to take up nutrients
What is clathrin-mediated endocytosis?
Target binds to receptor in clathrin coated pit - enters as coated vesicle - becomes uncoated and fuses with endosome.
Vesicles bud off endosome to return receptors to membrane
What is the clathrin triskeleton?
Consists of 3 heavy and 3 light chains - polymerise into lattice which forms the pits
What is dynamin?
Needed to pinch off clathrin coated vesicles
What is key for the function of the endocytic pathway?
The gradient of pH
What does cargo for degregation get packaged in?
Intraluminal vesicles (found in the multivesicular body)
What gene overexpression causes an enlarged endosome?
Rab5
How are yeast cells taken up by phagocytosis?
By dictyostelium
What are rab proteins?
Define intracellular organelles - in the endocytic pathway
What do lysosomes contain?
Hydrolases and lipases
How do we prepare competent cells for transformation?
We wash then with two buffers: TFB1 and TFB2 which contain rubidium and calcium - this induces permeabilisation
We then transform by hear shock
We then do antibiotic selection
When we transform cells, what controls do we do?
Tube with water - negative control
Tube with vector and ligase - controls for self-ligation
Tube with vector and insert
Tube with vector - positive control
GO OVER PRACTICALS
Why do we add phosphatase to vectors?
It reduces self-ligation of the vector to itself
What are the guidelines for primer design?
18-28 nucleotides in length
GC composition should be 50-60%
The melting temp should be 60-65% (4 x(G+C) + 2x(A+T))
What are the different types of plasmid species?
Slowest in gel electro: open circular
Linear
Fastest in gel electro: closed circular(supercoiled)
Small plasmids are usually supercoiled
What is a miniprep?
To isolate recombinant DNA from E.coli
Most commonly alkaline lysis - causes denaturing of DNA which reanneal after neutralisation - plasmids reanneal - genomic DNA is too big and gets tangled - easy to remove
What does the mitochondria do?
Makes ATP and has a key role in apoptosis
What is the mitochondria structure?
1-2 micrometres long
Double membrane
Inner membrane highly folded - cristae - contains REDOX proteins
Inner matrix has enzymes responsible for energy production, tRNAs, enzymes, DNA, ribosomes
Outer membrane has porins allowing entry of molecules <5000 kDa and contains enzymes involved in mitochondrial lipid synthesis
Intermembrane space has enzymes that use ATP to phosphorylate other nucleotides - H+ pumped in here
What is mitochondrial genetics?
Circular double stranded - ~ 15-17 kbps
Encodes for 37 genes
Inherited from mother
What is the mitochondria life cycle?
In a dynamic flux between fission and fusion
Fission - splits into 2 - 1 is good, 1 has debris so goes through mitophagy
Fusion - 2 into 1
Mitophagy - digested by a lysosome by encasing it in an autophagosome
How do we get proteins into the mitochondrial matrix?
Proteins N-terminal signal sequence is recognised by proteins embedded in membrane called TOMs - translocators of the outer membrane
The protein translocates through TOM then TIM23 (translocator of inner membrane) into the matrix
Signal is then cleaved off
How do we stop proteins folding before entering mitochondria?
Bind interacting proteins to chain - chaperones
Needs energy to dissociate
The signal sequence is +ve so the H+ gradient drives it through the IM
How do we get proteins into the mitochondrial outer membrane?
Major protein is porin - beta barrels
TOM can’t insert them - SAM (sorting and assembly machinery) inserts them and folds the protein
What are the two common routes to get proteins into the mitochondrial inner membrane?
1) TOM then TIM23
2) Protein completely enters matrix - signal sequence cleavage unmasks a 2nd signal causing insertion into OXA complex
if multipass - snake through TOM as a loop - allows chaperones to bind to stop folding - guide to TIM22
What is a peroxisome?
Single membrane - no DNA or ribosomes
Found in all eukaryotic cells - carry out oxidative reactions - have catalase and urate oxidase
Remove hydrogen atoms and make hydrogen peroxide: RH2 + O2 –> R + H2O2 - peroxidases use the peroxide
Used in alcohol and fatty acids metabolism
Where do peroxisomal membrane proteins come from?
Most made in cytosol and inserted in the membrane of preexisting peroxisomes - by peroxins - proteins don’t need to be unfolded
Pex5 recognises signal sequences (ser-lys-leu) and accompanies cargo into peroxisome - ubiquilated then recycled back
New peroxisomes arise from preexisting ones - fission
What translocation proteins are in the mitochondria?
Outer mitochondrial membrane:
- TOM (Translocator of Outer Membrane)
- SAM (Sorting and Assembly Machinery)
Inner mitochondrial membrane:
- TIM23/22 (Translocator of inner membrane)
- OXA (Cytochrome OXidase Activity)
What are microtubules?
Hollow tubes of alpha + beta tubulin
Has 13 protofilaments
Make cilia and flagella used to move sperm and respiratory fluids
What is the cilia and flagella structure?
Same in both just different lengths - flagella longer
Has a axoneme MADE OF MICROTUBULES:
- 9 doublets +2 microtubule assembly (9 doublets on the outside, 2 in the middle)
- has radical spokes (holds outer and inner together)
- has dynein arms (outer and inner of the 9 doublets) - motor protein which allows sliding - causes polarity as they touch one doublet but there isn’t any on the other side of doublet
- the rings look like they overlap - the full ring is complete (A) fibres - 13 protofilaments, the half ring is incomplete (B) fibres - 10 protofilaments
What do the different dynein arms do?
Inner - waveform
Outer - power
Cilia and flagella have different waveforms
What are nexin crosslinkers?
Anchor doublets so don’t slide past eachother - dynein will cause bending - not elongation
What are basal bodies?
At beginning of axoneme
9 x 3 (triplet) microtubule array
0.2 micrometre (diameter) x 0.4 micrometres (height)
There is no inner pair
What is the actin filament structure?
Has a plus and minus end (polarity) - plus end will be polymerised, minus is degraded
Globular with energy source in middle (ATP)
What are the differences between actin filaments and microtubules?
Microtubules have a bigger diameter (24nm), actin is 5-9nm
Microtubules are hollow (actin solid)
Microtubules are 50kDa, actin is 42kDa
Microtubule has a/b heterodimer
Microtubule has 450 amino acids, actin has 375
Microtubules have GTP (GTP in alpha, GDP in beta), actin has ATP (ADP in filament)
What are types of actin-binding proteins?
Monomer nucleating
Monomer sequestering
Capping
Cross-linking
Bundling
Depolymerizing
Membrane binding
What are the filaments and motors for cilia/flagella/cytoskeleton/muscle?
Cilia/flagella - filament = microtubule, motor = dynein
Cytoskeleton/muscle - filament = actin, motor = myosin
What is myosin’s structure?
Coil of 2 alpha helices
What is actin based motility?
Filopodium - small projections so cell can test environment
Lamellipodium - meshwork of filaments - extends cell membrane
Stress fibres - strong fibres which pull cell
Cortical actin
What does the extracellular matrix regulate?
Migration
Tissue integrity and cell shape
Proliferation
Differentiation
What is in the extracellular matrix?
Extracellular meshwork of proteins and hydrated macromolecules
Different types:
Basal lamina
Collagen and Elastic fibres
Proteoglycans and
Glycoproteins
What are the types of ECM?
Fibrous proteins - collagens and elastin
Adhesion proteins - fibronectin and laminin
Hydrated macromolecules - glycosaminoglycans and proteoglycans
What is the main ECM component?
Collagen
What is collagen’s structure?
Glycine, proline, hydroxyproline repeats
Triple helix - 3 alpha chains
Produced by fibroblasts and epithelial cells
1.5 nm wide
What is one collagen defect?
Ehlers-danlos Syndrome
Can pull skin as there is less collagen - less strength - bad in the heart
How is collagen synthesised?
1) synthesis of pro-alpha chain
2) hydroxylation of some prolines - allows crosslinking
3) glycosylation of some hydroxyprolines
4) 3 pro-alpha chains assemble
5) procollagen triple helix formation
6) secretion
7) propeptides are cleaved
8) self-assembly into fibril
9) clusters of collagen fibrils form a collagen fibre
What does cell motility need?
Energy, guidance, mechanical interaction with something outside, swimming/crawling, microtubules and microfilaments
What are glycosaminoglycans?
Disaccharide chains - 70-200 units long
Holds water
Highly charged
Proteoglycan - 95% sugar (80 saccharides)
Glycoprotein - 60% sugar (15 saccharides)
What are hyaluronan complexes?
Big protein with aggrecans attached - effective for holding water
What is laminin?
An adhesion glycoprotein
Self assembles
It has binding sites for integrins - allows to adhere to cells
What is fibronectin?
An adhesion glycoprotein
Self associates
Can bind to collagen and the cell
What are integrins?
Bind matrix through divalent cations
Removal of cations - cells detach
Binds ECM to cells
What are focal adhesions?
Is a transmembrane receptor - connects to cytoskeleton and integrins
Used for signalling
Contains FAK, Paxillin, Talin and Vinculin
What are the different focal adhesion knockouts?
B1 integrin - embryonic lethal day 5
a5 integrin - embryonic lethal day 10
fibronectin - embryonic lethal day 9
talin - embryonic lethal day 6-8
How is elastin made?
Tropoelastin —-> elastin
Need lysyl oxidase
What does actin do?
Moves organisms or cells
What is the myosin power stroke?
ATP powers the stroke
Calcium binds to troponin/tropomyosin so myosin can bind to actin
What are some integrin related defects?
causes bleeding gums/nose bleeds
What are adherens junctions?
Have cadherins - links to another cadherin - binds in cell to actin cytoskeleton
Calcium dependant
Homophilic interaction - cadherin binds to cadherin
What are the two actin-linked junctions?
Adherens junctions
Focal adhesion
What is the adhesion belt?
Cadherin -> catenin -> actin -> myosin
What is a desmosome and a hemidesmosome?
Hemidesmosomes - attach cells to basal lamina between integrins and intermediate filaments
Desmosomes - cell-cell junction between cadherins and intermediate filaments
What is pemphigus?
Autoimmune disease where immune system produces antibodies which attack desmoglein (cadherin which hold keratinocytes in the epidermis) - causes blistering
What are tight junctions?
Known as occluding junctions or zonulae occludens
Found in epithelia
Prevents fluid, ion and membrane flow, allows transcellular and paracellular transport, specialises membrane regions - apical (glycolipid and cholesterol), basal (phosphatidylcholine)
Sealing strands of claudin and occludin
What are gap junctions?
Is a connexon made of connexins (6 subunits can be hetero/homotypic) - form open channel
100-500nm long, 2-4nm gap between cells, 1.5 nm pore
Allows cell-cell communication
Regulates pH, Ca, membrane potential and cell signals
In connective tissue, heart, neurones ect.
What are selectins?
Slows down leukocytes when there is injury
Why do we incorporate restriction sites into primers?
So we can isolate our specific sequence
Add primers - forward attaches to start of gene, reverse to end of our gene
We can add sequences to the 5’ end of our primers - restriction site sequences
This allows for us to have sticky ends - no treatment needed
What stimulates cells to proliferate?
Extrinsic factors are needed - some other signals can override this and stop division
There is a master governor making the major decision regarding cell fate = cell cycle clock in nucleus
What are the 4 phases of the cell cycle?
M phase - mitosis: PPMAT and cytokinesis
Gap 1 - cells increase in size, ribosomes and RNA are produced and the cell is prepared for cell division
S phase - DNA replication
Gap 2 - cell checks DNA and is preparing for division
What checkpoints are there in cell cycle?
G1 - checks for DNA damage and favourable environmental conditions
S - check DNA for damage
G2 - check for damaged or unduplicated DNA
M - check for chromosome attachment
How long is each phase of the cell cycle?
G1 - 10 hours
S - 7.5 hours
G2 - 3.5 hours
M - 1 hour
- 22 hours
When does the cell decide to go to S phase?
Cell will test the environment from G1 to an hour or two before S - has until the R point (restriction)
E.g. cell has serum and growth factors removed 80% into G1 - went back to G0, when removed one hour before - went to S/G2/M phase
What deregulation accompanies the formation of most cancers?
Deregulation of the R-point
What ways can we model the cell cycle?
Genetic approach - requires cells that have a mutation in a potential cell cycle gene
Biochemical approach - requires large amount of cells in same transition state
What do we use to model the cell cycle?
Yeast
This is because:
- rapid division rate
- cell cycle control genes are highly conserved
- can be group as haploids or diploids
- easy to grow
How can we study genes which are crucial for cell survival?
We can grow diploid cells to maintain lethal mutation then study them haploid
There are temp sensitive mutations - allow growth in permissive temps (Cdc genes)
Can we use embryonic cells to study cell cycle regulators?
Yes!
We sucked out the cytoplasm from a cell in M phase and put it in a cell arrested in G2 - caused cell to go to M phase. Something catalysed this transition.
It was called maturation promoting factor
What controls a cell cycle transition?
A protein kinase-based machine (AA sequence of CDK - cyclase dependant kinase) - regulated by cyclins and Tyr phosphorylation
How do we visualise and quantify kinase activity?
Selective extraction of kinase - incubate with protein substrate and ATP - electrophoresis of substrate and imaging
How are cyclins involved in the cell cycle?
Cell cycle transition involves the irreversible destruction of cyclins
Cyclins activate kinases
What is the cell cycle regulator kinase gene in yeast?
Cdk1 - mammals have multiple as well as multiple cyclins
How do cyclins levels change throughout the cell cycle?
Cyclin E - low levels in G1 until R point - rapid increase
Cyclin A - levels increase as S phase
Cyclin B - levels increase in M
Collapse of cyclin levels - degration (ubiquitination dependant)
LOOK AT GRAPHS - lecture 25, slides 28/29
What are each cyclin’s partner?
G1 - Cyclin D - Cdk4, Cdk6
G1/S - Cyclin E - Cdk 2
S - Cyclin A - Cdk2 Cdk 1
M - Cyclin B - Cdk 1
What is cell-free mitosis?
We can use cell-free mitosis - can use a cytoplasm and remove it at different stages to study changes
What are the two phases of mitosis?
Chromosome condensation
Sister-chromatid resolution
What are homologous chromosomes?
Have the same genes arranged in the same order - one from mum, one from dad
What are chromatids?
Newly copied DNA strands joined by a centromere
Which Cdk drives entry to mitosis?
M-Cdk - cyclin B
Cdc25 phophatase removes an inhibitory cyclase from M-Cdk then activates a positive feedback loop - activated by S-Cdk
- triggers assembly of mitotic spindle
- chromosome condensation
- breakdown of nuclear envelope
- rearrangement of actin cytoskeleton and golgi
Wee1 inhibits M-Cdk but S-Cdk inhibits it
What is the anaphase-promoting complex?
Progression through metaphase and anaphase is driven by protein destruction - cyclin levels are zero
The APC is a ubiquitin ligase activated by Cdc20
1) Targets S+M cyclins - destroyed which unactivates most CDKs and CDK targets are dephosphorylated
APC/C is kept on in G1 - turned off as G1/S-CDK activated so cyclins can accumulate
2) Targets securin - protects protein linkages keeping sister chromatids together - destroyed so sister chromatids separate - anaphase
What can go wrong in mitosis?
Loss of heterozygosity - genes need mutations on both alleles to cause phenotypic change - 2 hit hypothesis e.g. 2 sporadic or one familial and one sporadic
Hemizygosity - loss of an allele
What is chromosome non-dysfunction?
Chromosomes end in wrong daughter cell
What is the structure of the mitotic spindle?
Interpolar microtubules - overlap
Kinetocore microtubules - attach to kinetochores (centromeres) - trial and error and the appropriate attachment is sensed by tension from chromatids
Astral microtubules - contact cell cortex to position spindle
Centrosome - centriole surrounded by pericentriolar matrix - nucleate microtubules
What does the destruction of securin cause?
Bound to an inactive separase - no phosphorylation to it as cyclins are destroyed and Cdks are inactivated - these would inhibit it but the APC stops this - the APC/C destroys securin
This activates separase - breaks sister chromatids apart
What is loss of heterozygosity by dysfunction?
Chromosome in wrong cell - elimination by apoptosis.
Sometimes the cell can eliminate a chromosome - sometimes it’s normal, sometimes it will cause loss of heterozygosity
What is loss of heterozygosity by mitotic recombination?
In G2/M - crossing over could occur - a chance that one cell gets both mitotic copies of a mutation.
What is loss of heterozygosity by gene conversion?
DNA polymerase begins replication on a template strand but then jumps to a different chromosome then back to normal.
This may pass a mutation to a different chromosome
How do we analyse proteins?
Levels: Western blot
Localisation: Immunofluorescence fusion proteins
Interactions: Pulldown assay, immunoprecipitation, yeast two-hybrid, ChIP (DNA-protein)
Methodology: Antibody, fusion protein
How do we analyse RNA?
Levels: quantitative RT-PCR, northern blot, microarray, luciferase assay
Localisation: FISH
Methodology: PCR, hybridisation, reporter genes
How do we analyse DNA?
Localisation: FISH
Interactions: ChIP (DNA-protein)
Methodology: PCR, hybridisation
What is meiosis?
Forms haploids - gametes
One homologue for each chromosome is in the gamete
2 steps: meiosis I and II
Ovary: oogonium –> primary oocyte –> secondary oocyte –> mature egg
Testes: spermatogonium –> primary spermatocytes –> secondary spermatocytes –> spermatids
How does meiosis cause genetic variation?
Crossing over and independant assortment
What happens in meiosis I?
Centrioles and chromosomes are replicated (like mitosis)
Maternal and paternal homologs pair up
One complete chromosome (2 chromatids) pulled to separate poles
Crossing over occurs
What is meiosis II?
like mitosis just makes haploid cells
What happens in prophase I?
Pairing - facilitated by synaptonemal complex
It aligns then for anaphase
Allows genetic recombination between paternal and maternal DNA
Can takes years to complete (mitosis is less that 30 minutes)
What is the synaptonemal?
Chromosome homolog pairing - brought 400nm apart - recombination complex binds them together
Axial core (binds chromatin via cohesion) are crossed liked by transverse filaments to form synaptonemal complex
This aligns the two chromosomes and helps crossing over (recombination)
What does homolog segregation depend on?
- both kinetichores attach to spindle pole - by protein complex which is removed after meiosis I
- crossing over
- cohesin is only removed from arms - keeps chromosomes together
How does crossing over happen?
At least one crossing over - no more than 4
Regulation makes sure there’s at least one and inhibits others close by - crossover interference
What are the two categories of chromosome abnormalities?
i) abnormalities in chromosome number
ii) chromosome structural rearrangements
What is aneuploidy?
Different numbers of chromosomes - non-dysfunction
Monosomy - 1 copy of a chromosome - lethal
Trisomy - 3 copies - usually lethal
Polyploidy - extra sets e.g. triploid - lethal
Can have an extra sex chromosome - normal lifespan
One sex chromosome - X - normal but infertile, Y - not viable
Caused by failed separation in Meiosis I/II or mitosis - either from sister chromatids or homologous chromosomes
What is trisomy 18?
Severe intellectual disability
Low birth weight
A small, abnormally shaped head
A small jaw and mouth
Clenched fists with overlapping fingers
Congenital heart defects
Various abnormalities of other organs
Most die before 1 month
- edward’s syndrome
What is trisomy 22?
Undeveloped midface
Malformed ears
Wide-spaced eyes
Microcephaly
Congenital heart disease
Usually die shortly after birth
What is 45, XO turner’s syndrome?
Complete or partial absence of a second sex chromosome in females
- poor growth
- short
- delayed puberty
- congenital heart defects
- skeletal abnormalities
1% survival rate as there is haplodeficiency (pseudoautosomal genes need to be expressed in both alleles) or imprinted genes on X
When do structural rearrangements occur?
Usually in homologous recombination or DNA damage - represent 4.7% of 1st trimester abnormalities
Can view them via spectral karyotyping (SKY)
How do cells die?
Mostly through necrosis or apoptosis
When does necrosis occur?
Physical damage - trauma (cuts/burns) and extreme temperature
Toxins - external (snake venom), internal (bacteria)
Acute hypoxia/ischaemia - stroke
When does apoptosis occur?
Physiological:
- tissue size maintenance
- development
- removal of immune cells
- hormone-dependant involution
- inappropriate interactions e.g. anoikis
Pathological:
- DNA damage
- viral infection
50 billion cells daily
What are the characteristics of necrosis?
Reversible: membrane collapses, organelle and cell swelling
Irreversible: increased intracellular calcium, autolysis, cell bursting, makes an inflammatory response
Causes blebbing - cells leak into extracellular fluid
No ATP required
What are the characteristics of apoptosis?
Shrinkage
Nuclear breakdown
Apoptotic bodies - budding
Phagocytosis
No inflammatory response
Requires energy
Controlled cell death
Capsase is mediator
How do cells die in brain ischaemia?
Cells in middle die - necrosis
Cells at edge die - apoptosis
This restricts spread of cell death
What is developmental apoptosis?
Metamorphosis: e.g. frog
Thyroid hormone in blood causes apoptosis in tails
Digit formation in mice cause by local signal proteins
Neuronal connections are refined by the competition for survival factors - not enough factors = apoptosis
What are ced genes?
C-elegans - good model to study apoptosis
Some Ced genes can recognise the apoptotic signal and some cause phagocytosis of the cells
Many of these genes are conserved:
EGL-1 - BH3-only proteins
Ced 9 - Bcl 2
Ced 4 - APAF-1
Ced 3 - capsases
Reduced Ced 3+4 - gives excess adult cells
Reduced Ced 9 - massive cell death
EGL-1 stop CED 9
CED 9 stops CED 4
CED 4 promotes CED 3 - then cell death
What are caspases?
Causes apoptosis
C = cysteine in active site
asp = aspartic acid cleave target proteins
Irreversible
Over 10 Ced3 homologues
What are the types of capsases?
Initiator: activated by apoptosis signals and activate executioner capsases
Executioner: cleave over 1000 proteins
Amplifying proteolytic cascade: one initiator can activate multiple executioner
What are some caspase targets?
They can cause nuclear breakdown including the nuclear lamina
They can prevent DNA repair by cleaving PARP
They can cause cytoskeleton changes e.g. breaking down actin
They cleave proteins that inhibit apoptosis, DNA repair, cell cycle and nuclear structure
What are the two pathways which activate apoptosis?
Extrinsic
Intrinsic
What is the apoptosis extrinsic pathway?
By tumour necrosis factor family are ligands for death receptors
6 receptors: death receptors which indirectly activate initiator capsases by DISC
e.g. Fas ligand on killer lymphocyte –> capsase 8 (initiator), DISC is made = death-induced signalling complex
What is the intrinsic apoptosis pathway?
Triggered by: stress & development signals
e.g. cytochrome c is released by mitochondria, Apaf1 is activated –> apoptosome is assembled –> caspase 9 is recruited (initiator) –> activates executioner capsases
LOOK AT SUMMARY ON LECTURE 28, slide 22
What is the pro and anti apoptotic factors?
Bcl2 family proteins:
EGL-1 - BH3-only protein is pro
Ced 9 - Bcl2 protein is anti
There is a balance of each
What is cancer?
A disease of aberrant cell proliferation and differentiation
e.g. tumours have no layers compared to normal cells which are arranged in layers
Does cancer occur in similar frequencies in different populations?
No - some countries have a high risk for certain cancers and some have a lower risk
There was two migrations of japanese people to hawaii 50 years apart - we can look at the cancer rates.
Japanese - high stomach, low prostate/breast/prostate
Caucasion - higher for all cancers
Hawaiian Japanese - just below caucasion
Shows that environment can dictate the cancers
How does infection cause cancer?
Infection - a chicken with breast cancer had it’s tumour grinded up and passed through a filter - then injected the filtrate into a healthy chicken - got a tumour - shows cancer can be caused by infection
DNA contains a gene called Src (Oncogene) which controls pathways that control cell adhesion, proliferation and cell mobility - viruses make a hyperactive version of this tyrosine kinase gene which turns on all the pathways
e.g.
Nasopharyngeal cancer - epstein-barr virus
Cervical - HPV
Kasposi’s - human herpesvirus 8
How does diet cause cancer?
Aspergillus oryzae (Koji mold - rice, peanuts) - increases risk of liver cancer
This is because when cytochrome P-450 modifies aflatoxin to aflatoxin-2,3-epoxide - this can bind to a guanine in DNA - mutated base
How does noxious substances cause cancer?
E.g. asbestos (naturally occurring silicate) - this can cause pleura mesothelioma
What environmental influences cause cancer?
Mostly due to smoking - 33%
Diet/obesity - 25%
Viruses - 5%
UV - 2%
Genetic factors are also causing
What are some cancers not due to environment?
Retinoblastoma
Li-Fraumeni syndrome
Wilm’s tumour
Gorlin’s syndrome
Breast cancer syndrome
What is an oncogene?
A gene with the potential to cause cancer by transforming cellular behaviour - they are dominant
e.g. SCR
Mostly arise from genes involved in regulated proliferation - Proto oncogenes
These come about from chromosomal rearrangement (hyperactive), gene amplification (protein made loads), regulatory mutation (protein made loads) or a deletion/point mutation (hyperactive protein)
How do chromosomes relate to CML (chronic lumoid leukaemia)?
Chromosomal changes cause cancer e.g. CML patients had different chromosomes: 22 and 9 due to translocation of some chromosome 9 to 22 and vice versa
This is due to a fusion of genes ABL (positive regulator of cell growth) and BCR - ABL cannot turn itself off now
FISH allows us to identify bits of DNA complimentary to what we add - we can see that chromosome 9 parts are in the wrong place
Do viruses contain an oncogene?
Most likely as viruses benefit greater from dividing cells
What was the first identified oncogene?
Ras - GTPase turns it off, exchange protein turns on
When bound to GTP = active
When bound to GDP = inactive
When a growth factor binds to a TK receptor - phosphorylated Grb2 and Sos - Sos will activate Ras by removing GDP allowing Ras to add GTP - growth factor induced growth
Glu61, Gly12 are critical for turning on and off - mutations make it go from a proto-oncogene to an oncogene
How is Ras important in growth factor induced growth?
Causes cell growth
Causes gene expression
Causes cell morphology and movement
Mutated Ras - cause tumours as can’t turn off
What is the cell fusion experiment showing how dominant oncogenes cannot explain cancer cell behaviour?
Cell fusion - a normal cell and cancer cell fusion
The cell put into mouse - normal healthy mouse
There is something dominant in normal cells which will suppress the oncogene
This implied the existence of tumour suppressor genes
What provided insight into tumour suppression?
Retinoblastoma
Arises sporadically (in one eye) or familial (both eyes)
When you plotted one eye vs both eyes - both eyes was a straight line showing only one thing needs to change (one hit meaning they had one inherited mutated gene and another happened randomly), one eye was a curve showing two things need to change (2 random mutations)
This one/two hit hypothesis provides evidence for tumour suppression, that cancer requires loss of both wild types and the basis of inherited cancers
What are some tumour supressor genes in familial cancer syndromes?
Retinoblastoma - Rb
Li-Fraumeni syndrome - P53
Wilm’s tumour - WT-1
Gorlin’s syndrome - Ptc
Breast cancer - BRCA-1
FAP - APC
What is the difference between tumour suppressor genes and oncogenes?
Oncogenes are activating, gain of function and are dominant. Only one allele mutation is needed
Tumour suppressor genes are inactivating, loss of function and recessive. Need two mutated alleles
How many mutations are needed for cancer?
More than one
Incidence rate and age is a curve - showing it cannot be one mutation as it would be a straight line
Higher number - nastier cancer
How can we see cancer in chromosomes?
Chromosome painting - we can see translocations
Cancer genome sequencing - we can see where things have moved compared to normal
What are causes of genetically instability?
DNA repair pathway defects
Defects in correction mechanisms in DNA replication
Defects in correction mechanisms for DNA segregation
What contributes to tumourigenesis?
Increased cell division and decreased apoptosis
How do normal cells respond to cellular stresses?
E.g. DNA damage, telomere shortening, hypoxia
Stable p53 will cause cell cycle arrest, senescence and apoptosis - cell cycle checkpoint
p53 is mutated in almost all cancers - disrupt intrinsic apoptosis
Mutated cell cycle checkpoints usually cause cancer e.g. Rb which operates in the restriction point
What is the structure of cholesterol?
Polar head group with nonpolar hydrocarbon tail
What determines blood group?
The structure of the oligosaccharides attached to sphingomyelin
O - no terminal sugar
A - N-acetylgalactosamine (GalNAc)
B - galactose
What ion is transport driven by?
Mammalian membrane - Na+
Bacteria/yeast - H+
What helps new proteins fold?
chaperone proteins
What vesicles do cargo for degradation go into?
Intraluminal vesicles
Late endosome: multivesicular body
How long are the cilia and flagella?
Cilia: 2-10 x 0.25 micrometres
Flagella: 100-200 x 0.25 micrometres
What are cyclins controlled by?
D type - Extracellular signals
All - CDK inhibitors (CDI)
What is active transport mediated by?
Coupled carrier
ATP driven pump
light driven pump
