MGD Flashcards
What are the functions of the Golgi Body?
Export of proteins
Membrane synthesis
Detoxification reactions
What is the function of the cytoplasm?
Metabolism of carbohydrate, amino acids and lipids
Fatty acid synthesis
What is the function of lysosomes?
Cellular digestion with hydrolytic enzymes.
What are the functions of mitochondria?
ATP synthesis
Beta oxidation of fatty acids
What is the function of the endoplasmic reticulum?
Lipid and steroid synthesis (smooth), protein export. membrane synthesis, detoxification
What is the function of the nucleus?
DNA synthesis and repair. DNA is in the form of chromatin
What is the function of the nucleolus?
RNA synthesis and ribosome assembly
What is the function of the plasma membrane?
Transport of ions and small molecules, cell morphology and movement
What is the function of a ribosome?
Where are they found?
Protein synthesis (free in cytoplasm or attached to RER)
What is a bacterial cell wall made of?
Peptidoglycan
How many pairs of chromosomes are there in a human cell?
23
How is DNA present in a bacterial cell?
In a circular loop
What type of ribosomes are found in:
Eukaryotes?
Prokaryotes?
Eukaryotes: 80S
Prokaryotes: 70S
What does solubility depend on?
The ability to make hydrogen bonds
How are monomers joined together to form macromolecules?
Covalent bonds
How are macromolecular complexes held together?
Non-covalent interactions
What is a hydrogen bond?
An electromagnetic attraction between a hydrogen atom bonded to an electronegative atom, and another electronegative atom
Define the term hydrophilic:
Polar molecules which can interact with water forming hydrogen bonds, and therefore dissolve in water
Define the term hydrophobic
Non polar molecules which can’t interact with water molecules, and are therefore insoluble in water
What is a hydrophobic interaction?
Non polar regions/molecules cluster together in order to repel water.
What is an amphipathic molecule?
A molecule with both polar and non polar ends
What is pH?
A measure of the concentration of H+ ions in solution
-log[H+]
What are van der Waals forces?
forces between two atoms in close proximity.
How to strong and weak acids differ?
Strong acids dissociate completely in solution, whereas weak acids only partially dissociate.
What is Ka (and pK), and what do different values of it mean?
Ka is the acid dissociation constant, a measure of how strong or weak an acid is, -log to give pK. Strong acids have a large Ka (low pK), and weak acids have a small Ka (large pK).
What is a buffer?
A mixture of a weak acid and its conjugate base, which resists changes in pH on addition of a small amount of acid or base.
Henderson Hasselbach equation
pH = pKa + log [A-]/[HA]
When pH>pK…
The deprotonated form dominates
(Higher pH, less H+ in solution, so acid dissociates to give more)
When pH<pk...>
</pk...>
The protonated form dominates
(There is lots of H+ in solution so the acid is less likely to dissociate)
When pH = pK…
The amount of acid and its conjugate base is equal
How can amino acids be classified?
Aliphatic/aromatic
Non polar, polar uncharged, postive, negative
What are the 2 negatively charged amino acids?
Aspartate and glutamate
(acidic, lose a proton)
What are the 3 positively charged amino acids?
Lysine, aspargine, histidine
What is a peptide bond?
A covalent bond between the O of a Carboxyl group and the H of an amino group
What are 4 key features of peptide bonds?
All atoms in the bond are on the same plane.
Formed in a condensation reaction.
No rotation.
Trans orientation.
What is a zwitterion?
A neutral molecule with both a positive and negative charge
What is the optimum blood pH? and what range must it stay within?
7.4
Range: 7.38 to 7.42
Define pI
Isoelectric point: The pH value at which the protein has no overall charge.
Properties of acidic proteins
Lots of negatively charge amino acids
Low pI
Properties of basic proteins
Lots of postive amino acids
High pI
If pH is higher than isoelectric point…
NEGATIVE ion
If pH is lower than isoelectric point…
POSTIVE ion
Define primary structure of a protein
Bonds involved?
The linear amino acid sequence
Covalent
Define secondary structure of a protein
Bonds involved?
The local spatial arrangement of the polypeptide backbone.
Hydrogen bonds
Define the tertiary structure of a protein
Bonds involved?
The 3D arragement of all atoms in a polypeptide, the folding of secondary structures.
Hydrogen, ionic, disulphide, hydrophobic interactions, van der waals
Define quarternary structure of a protein
Bonds involved?
The 3D arrangement of multi sub unit proteins, with more than one polypeptide chain.
Same as tertiary
What are the major features of an alpha helix?
RIght handed helix
- 6aa per turn
- 54nm pitch
Carboxyl of one aa bonded to amine 4 aas away
Ala and Leu, small hyrdophobic aas, strong helix formers
Gly and Pro are helix breakers
R groups point outwards
What are the key features of a beta pleated sheet?
Extended conformation
0.35nm between adjacent aas
Parallel/antiparalell
Alternate side chains in opposite directions
B strands make B sheets
What factors cause protein denaturation? Explain
Heat: increased vibrational energy.
pH: alters ionisation states of aas.
Detergents/ organic solvents: disrupts hydrophobic interactions, breaking tertiary structure
What is an amyloidoses?
Altered conformation of a protein promotes conversion of an existing protein into a misfolded one, forms insoluble aggregates.
Usually highly ordered with large proportion of beta sheets.
Inter chain assembley stabilised by hydrophobic interactions.
Stain: CONGO RED
What is a fibrous protein?
Only primary and secondary structure
Insoluble
Support function
No tertiary
Single type of repeating secondary
Long strands/sheets
Collagen
What is a globular protein?
Up to quaternary structure
Soluble
Catalysis and regulation
Compact
Several types of secondary
Lots of tertiary
Explain the physiological role of myoglobin
Single subunit protein
One Haem group
So can bind one oxygen molecule
Stored in the muscles, used when oxygen is in high demand, acts as a store
Hperbolic oxygen binding
No cooperativity
Explain the physiological role of haemoglobin
Tatrameric protein
2 alpha and 2 beta subunits
Four Haem groups
4 molecules of oxygen
Sigmoidal/cooperative binding
Picks oxygen up in the lungs and delivers it to the tissues for respiration
Some carbon dioxide transport
Why does oxygen need to be transported by proteins?
Non polar
Can not diffuse far in water
Describe the affinity of myoglobin for oxygen
Very high affinity for oxygen
Will only release oxygen with pO2 is very low
Hyperbolic curve
No cooperativity
Describe the oxygen binding properties of haemoglobin
Cooperative binding
Sigmoidal binding curve
Low affinity T state = tense
High affinity R state = relaxed
Transition between the two gives the sigmoidal curve
Describe the properties of the haem groups
Porphyrin ring and an Fe atom bound to 4 N atoms of the ring
Bound to protein via histidine residue
Fe sits slightly below the ring, O2 binding causes it to move into plane
Changes the conformation of the bound polypeptide
What is cooperativity in Haemoglobin?
Low affinity T state and high affinity R state
Oxygen binding changes the conformation in all 4 sub units, promoting the binding of subsequent O2 molecules
The sigmoidal curve makes it a more efficient carrier, more sensitive to smal differences in pO2.
What is the effect of 2,3-bisphosphoglycerate on the binding of oxygen to Hb?
Regulates oxygen binding
Binds to Hb
Decreases oxygen affinity
Curve shifts to the right
1 BPG per tatramer of Hb
[BPG] increases at high altitudes promoting O2 release to tissues
A BPG isomer is produced in glycolysis, so O2 is released more readily in areas that are metabolically active
What is the effect of carbon dioxide and H+ ions on the affinity of Hb for O2?
Both bind to haemoglobin
More acidic
Lowers the affinity of Hb for oxygen
Sites of low pH and high carbon dioxide
BOHR EFFECT
Curves shifts to the right
Release of oxygen
Metabolically active tissues produce lots of H+ and CO2
Ensures delivery of oxygen is coupled to demand
What is the effect of CO on the binding of oxygen to Hb?
Binds tightly to Hb, 250x more readily than oxygen
Prevents O2 binding
Blocks O2 transport
Fatal when COHb is more than 50%
Increases the affinity of unaffected sub units for oxygen
High affinity state: releases less oxygen to tissues
What inheritance pattern does sickle cell anaemia have?
What mutation causes it?
Autosomal recessive
Single nucleotide subsitution
Missense
A changes to a T
Glutamate to Valine
Beta subunit
What effect does the mutation in sickle cell anaemia have?
Blood disorder
Valine creates a hydrophobic pocket in the beta sub unit
Causes deoxygenated Hbb to polymerise
Distortion of RBCs into sickle cell shape
Stress to cell membrane, premature cell lysis
Lifespan of only 30 days compared to 120 days
Blocks microvasculature
What are thalassaemias?
Group of haemoglobinopathies
Genetic disorders
Imbalance between alpha and beta subunits
What is alpha thalassaemia?
Descreased or absent alpha chains
Different severity levels as multiple copies of alpha gene are present
Beta chains can form stable tetramers with increased affinity for oxygen
Onset before birth
(as alpha subunits in fetal Hb a2y2)
What is beta thalassaemia?
Decreased or absent beta chains
Excess alpha chains
Can’t form stable tetramers
Symptoms appear after birth
As B globin only in adult Hb a2B2
3 - 6 months after birth, lose fetal Hb
Explain the effects of enzymes on chemical reactions?
Lower activation energy needed for a reaction to occur
Bidning of substrate to an active site
Increases local concentration of reactions
Stabilised formation of a high energy transition state
Increase rate of reaction
Non convalent binding of substrate, highly specific
Describe some properties of enzymes
Do not effect the equilibrium of the reaction
Proteins
Some require cofactors: inorganic ions
Some need coenzymes: organic carriers of reaction groups
Highly specific
Unchanged after reaction
Active site: a few amino acids supported by a scaffold, cleft or crevices, to exclude water
Substrate changes enzyme shape slightly, induced fit
How does reaction rate vary as a function of enzyme and substrate concentration?
An enzyme E, combines with a substrate S, to make an enzyme substrate complex, ES, to break down to form free enzyme and product, P.
From this can derive michaelis menten equation, which describes how reaction velocity varies with substrate concentration
Increase substrate concentration, reaches maximal velocity, rectangular hyperbola
Increase enzyme concentration, directly proportional to reaction rate
What is the international unit of enzyme activity?
Define Km
Define Vmax
Unit: the amount of enzyme that produces one micromole of product per minute
Km: the substrate concentration that with give half the maximal rate; Vmax. Also equates to affinity of enzyme for substrate. Low Km = high affinity and vice versa
Vmax: the maximum velocity/rate of an enzyme catalysed reaction, when the enzyme is saturated with substrate
Interpret kinetic data for enzyme catalyed reactions:
Vo:
Lineweaver Burk Plot:
How to interpret Km
How to interpret Vmax
Slope
Vo is the initial reaction velocity
Lineweaver Burk Plot:
X axis: 1/[S], intercept is Km
Y axis: 1/V, intercept is Vmax
Slope is Km/Vmax
Effects of inhibitors on enzyme kinetics:
No inhibitor:
Irreversible inhibitor:
Reversible inhibitors -
Competitive:
Non-competitive:
No inhibitor: reaches Vmax
irreversible: covalently bonded
Competitive: binds at active site, affects Km but not Vmax. Adding enough substrate will overcome effect.
Non competitive: binds away from active site, affects Vmax, but not Km, cannot be overcome
List the major regulatory mechanisms that control enzyme activity:
Allosteric: enzymes with more than one subunit, the binding of substrate to one site can inhibit or activate and enzyme. Cooperativity. Eg phosphofructokinase, activated by AMP, Fructose 2 6 bisphosphate. Inhibited by ATP, citrate, H+
Substrate and product concentration: substrate availability, co enzyme availability, accumulation of product can inhibit forward reaction, eg glucose 6 phosphate inhibits hexokinase
Covalent modification: attachment of groups covalently via amino acids. Attachement of phosphates via kinases and removal by phosphatases.
Proteolytic activation: secreted as inactive zymogen and cleaved by proteases to active enzyme, eg trypsinogen to trypsin
Changes in the amount of enzyme: regulation of enzyme synthesis and protein degradation
What is an enzyme cascade?
When enzymes activate other enzymes
The number of affected molecules increases in the enzyme cascade
Number of affected molecules increases geometrically
Allows quick amplification of an initial signal by several orders of magnitude quickly
Give examples of zymogens and their active enzymes
Also give their activators and where they are found
Pepsinogen to pepsin, activated by pH in the stomach
In the pancreas:
Trypsinogen to trypsin activated by enteropeptidase
Chymotrypsinogen to chymotrypsin activated by trypsin
Procarboxypeptidase to carboxypeptidase activated by trypsin
Proelastase to elastase activated by trypsin
a1 antitrypsin deficiency linked with emphysema
Describe allosteric enzymes
Multi sub unit enzymes that contain more than one active site
SIgmoidal relationship, cooperative
2 different comformations: tense and relaxed
Substrate binding to pme subunit makes progressive binding easier
PFK, step 3 of glycolysis:
Activators: AMP, fructose-2,6-bisphosphate
Inhibitors: ATP, citrate and H+
What do allosteric activators and inhibitors do?
Effect on curve
Activators: increase activity of enzyme, curve shifts to the left, increases proportion of enzyme in R state
Inhibitors: decrease activity of enzymes, curve shifts right, increases proportion of enzyme in the T state
T converts to R upon binding, equilibrium shifts to the right
What do kinases do?
Transfer the phosphate group from ATP
To the -OH group of Ser, Thr, Tyr
Big negative charge added
Changes the conformation, substrate binding
What do phosphatases do?
Removed phosphate groups through hydrolytic activity
Define the term zymogen
How are they activated?
What sort of enzymes are commonly made as zymogens?
Examples?
Inactive precursors of enzymes
Activated by removal of part of the polypeptide chain
Many proteases are synthesised in this way: makes for safe transport
Blood clotting factors
Apoptosis: procaspase to caspase
How can enzymes be regulated by changes in the amount of enzyme?
Change in transcription rate, ribosome rate
Regulated protein degradation, tag with the small protein ubiquitin
Explain how the activation of the clotting cascade leads to the formation of fibrin
Intrinsic pathway, damaged endothelial lining of blood vessels promotes binding of factor 11a
Extrinsic pathway: trauma releases factor 3, tissue factor
Postivie feedback from thrombin, further activation of cascade to allow clot formation from very small amount of initial factor.
Factor 10 bring both together, the 2 THROMBIN, then 1 FIBREIN, stabilised by calcium
What is the role of calcium and vitamin K in the clotting cascade?
Release of calcium
y carboxyglutamate, Gla domains, carboxylated on clotting factors IX and X
Need vit K as co factor
Target appropriate sites for activation
Activates factors
Cleaved proteolytically
Brings together clotting factors
Calcium needed for factors 1, 2, 10, 9 and 7
How is a clot formed?
Fibrinogen to fibrin
Fibrinogen: 2 sets of tripeptides a, B, y, joined at N terminal by disulphide bonds. N terminal regions highly negatively charged, prevent fibrinogen aggregation
Prothrombin binds Ca2+ on gla residues to thrombin
Thrombin cleaves fibrinogen A and B feet
C terminal ends of B and y (globular) interact with cleaved N terminal ends of A and B to form fibrin clot
Cross bridges between lysine and glutamine residues, catalysed by transglutaminase
What do these blood clotting factors do?
III?
VIII?
XI?
III: tissue factor, extrinsic pathway, trauma activates
VIII: haemophilia A
XI: intrinsic pathway
Describe regulation of the blood clotting cascade?
Inactive zymogens present at low concentrations, so to prevent accidental clot activation
Dilution of clotting factors by blood flow and removal by the liver
Amplification of signal
Localisation of clotting factors to site of damage: factor with Gla binds to endothelial cells which are damaged
Feedback activation by throbmin, enhances conversion of V, VIII and XI to active forms
Termination by many processes: Digestion of factors by proteases, Va and VIII a by protein C, activated by thrombin
Plasmin degrades clots. Converted from plasminogen by t-PA and streptokinase
Specific inhibitors antithrombin 3, enhanced by heparin binding, does not act on thrombomodulin bound thrombin (on endothelial cells)
What are the components of RNA and DNA molecules?
Nitrogenous bases
Pentose sugar
Phosphate, ester bond between it and C5 of sugar
What bases are present in DNA?
What bases are present in RNA
C, G, A, T
C, G, A, U
What are the types of nitrogenous base?
What is the difference between them?
Which bases are which type?
How are base pairs formed?
Purine and Pyrimidine
Purines have 2 rings, Pyrimidines have 1
A and G are purine. C, T and U are pyrimidine
Base pairs are formed between purines and pyrimidines, by hydrogen bonds
What is the charge of a DNA molecule? Why?
Negative
From phosphate group
What sugar is in RNA?
What sugar is in DNA?
Ribose
2-deoxyribose
How many strands in
RNA?
DNA?
RNA: 1
DNA: 2
What is a nucleoside?
What is a nucleotide?
N side, sugar and a base
N tide, sugar, base and phosphate
How are DNA and RNA sequences typically read?
5’ to 3’
5’ starts with phosphate, 3’ is hydroxyl
Top strand is always 5’ to 3’
Left to right
Bases given letters
Duplex structure include complementary antiparallel strand
Hydrogen bonds denoted by dotted lines
Explain the polarity of a DNA or RNA chain
Polynucleotides are nucleotides linked covalently by phosphodiester bonds
Each single strand has polarity
5’ end is free phosphate
3’ end is free OH
Which nitrogenous bases pair together?
How many bonds between each?
C and G, 3 H bonds
A and T/ U, 2 H bonds
How do the hydrogen bonds form between bases?
O atoms in carbon ring are elctronegative
N group is dipole positive
Interaction between electronegativity of oxygen and dipole of nitrogen
How are duplex structures formed in RNA and DNA?
How are RNA stem loops formed?
Complementary antiparallel strands
DNA-DNA/RNA-RNA/RNA-DNA
Stable/temporary/invivo/in lab
DNA double helix
RNA, strand loops back on itsself, one side is antiparallel, H bonds between complementary bases
Describe the key features of the DNA double helix
2 independent polymers
Complimentary and antiparallel
Top strand 5’ to 3’, vice versa
Space between base pairs 0.34 nm
Purines and pyrimidines planar and unsaturated
Major grooves of exposed bases
and minor grooves
in structure of sugar phosphate backbone
How is eukaryotic DNA condensed in chromosomes?
Chromatin is present in interphase
NUCLEOSOMES: beads on a string, DNA wound twice around histone core, which is postiively charged
This is EUCHROMATIN, expressed genes, can replicate in interphase
SOLENOIDS: Nucleosomes are coiled to form 30nm fibres, highly condensed, can not be replicated, mitotic chromosomes
What phase of interphase is DNA replicated in?
S
Describe the process of DNA replication
INITIATION:
Strands unzipped by HELICASE. Origin of replication recognised, each strand initiated by PRIMASE. Specific proteins must interact with DNA and recruit DNA polymerase (can only extend from 3’ ends)
ELONGATION:
Each strand replicated by DNA polymerase, 5’ to 3’. Leading strand, continuously. Lagging strand, discontinnously in Okazaki fragments. Fragments joined by DNA ligase from OH group to phosphate group covalently.
TERMINATION:
Replication forks join, leading moves towards lagging.
What sort of replication is DNA replication?
Semi-conservative
one of the original DNA strands is in each of the new molecules, in each of the daughter cells
How does DNA polymerase work?
Uses deoxyribonucleoside triphosphate molecules as substrate
Base sequence determined by complementary template strand
Driven by pyrophosphate hydrolysis
How do helicase enzymes work?
Use ATP energy to unwind the DNA helix
Allow DNA replication
Around the replication fork helicases reform the DNA helix
Multiple replication forks along dsDNA
Describe the process and role of the cell cycle
G1 phase: growth, cell content replication, preparation for replication
S: DNA replication
G2: double check chromosomes and repair
MITOSIS and CYTOKINESIS, used to maintain and repair the body
Several key checkpoints to prevent improper cell division
Outside: G0, cell cycle arrest, for example nerve cells. Some may go back to G1
Describe the process of mitosis
All genetic info replicated in the S phase of interphase
Prophase: breakdown of nuclear membrane, chromosomes condense, kinetochore of spindle fibres attaches to centromere
Metaphase: chromosomes in X shape line up on metaphase plate
Anaphase: centromeres divide, spindle fibres contract, sister chromatids to opposite poles
Telophase: nuclear membrane reforms, chromosomes decondense, spindle disappears, cleavage of cytoplasm, cytokinesis
How to chromosome exist before mitosis, after replication?
Identical sister chromatids, joined by centromere
X shape
2 p arms and 2 q arms
Telomere at end of chromosome arm, prevents degradation
Describe the role of mitosis
Diploid cell
1 round of replication, 1 round of division
Cell division for somatic cells
Needed for tissue growth and early development
Describe the role of meiosis
Diploid cell, one round of replication, 2 rounds of division
4 haploid cells
Specialised cell division for germ line cells
2n to n
Production of eggs and sperm
Maintains constant chromosome number, diploid zygote
Generates genetic diversity from crossing over in prophase 1, and random assortment in metaphase 1
Produces 4 sperm and 1 egg, with polar bodies
Describe the process of meiosis
Prophase 1: nuclear membrane disappear, chromosomes condense, bivalent forms, crossing over to form chiasmata
Metaphase 1: homologous pairs line up at metaphase plate randomly, bivalent. Independent assortment.
Anaphase 1: Homologous pairs pulled apart as spindle fibres contract. Nuclear membrane reforms.
Telophase 1: cytokinesis
Prophase 2: new spindle develops
Metaphase 2: chromosomes line up on metaphase plate
Anaphase 2: centromere divides, chromatids pulled to opposite ends by spindle
Telophase 2: chromatids lengthen and are indistinct, spindle disappears, nuclear membrane reforms, cytokinesis
How common are mistakes in meiosis?
1/3 of all miscarriages
What is genotype?
The genetic make up of an individual
Either as a whole
Or one specific genetic locus
Genes
What is a phenotype
All observable characteristics of an individual
Expressed trait
As a result of the genetic make up
Of one or more specific gene loci
Expressed proteins
Explain how environmental factors have an effect on both phenotype and genotype
For example
UV changes genotype to cause cancer
Unhealthy food changes phenotype, fat
Environmental factors:
Radiation
Mutagens
Chemicals that affect cell growth
Diet
Lifestyle
What is a gene?
A unit of heredity
A length of DNA on a chromosome that codes for a protein
What is an allele?
An alternative form of a gene
Each individual has two alleles of every gene
Can either be the same or different
What are the different patterns of inheritance?
AUTOSOMAL:
Dominant
Recessive
SEX LINKED:
X linked dominant
X linked recessive
Y linked
Explain autosomal dominant inheritance
Heterozygotes affected
Someone affected in every generation, all affected have an affected parent
Males and females equally affected
Rare disease in homozygotes: usually fatal
50% chance child is affected
Examples: huntingtons, neurofibromatosis 1, Marfan’s, familial hypercholesterolaemia IIa
Explain autosomal recessive inheritance
Homozygotes only affected
Can skip generations, can seem to come out of nowhere
Males and females equally affected
Heterozygotes unaffected, carriers
2 heterozygotes, 25% chance affected offspring
2 homozygotes, only affected offspring
Examples
Cystic fibrosis, sickle cell disease, Tay Sachs
Explain X linked recessive inheritance
Hemizygous males, always affected
Homozygous females affected rare
More common in males
Heterozygous female carrier, 50% chance of affected sons
Affected males can’t give trait to sons
Every affected male, heterozygous carrier mother
Every affected female, carrier mother, affected father
Daughters of affected males heterozygous
Examples: Haemophilia A, reg green colour blindness, Duchenne muscular dystrophy
Explain Y linked inheritance
Rare
When gene is located on Y chromosome
Inherited directly from father to son
Define dominance
Phenotypic trait is dominant when it occurs in heterozygotes and homozygotes
Define recessive
When a phenotypic trait is only expressive in the homozygote
Define Co dominance
When both allele in a pair are expressed and neither is dominant
Describe complementation
more than one gene can be involved in producing a phenotype
EG ALBINISM
Therefore a child of two affected parents can be unaffected if parents have different defective genes
a1a1A2A2 x A1A1a2a2
CHILD
a1A1a2A2
Describe the basis of co inheritance of certain traits
Genes on the same chromosome are linked, not independtly assorted at meiosis
Genes on different chromosomes are not linked, independently assorted in meiosis
If they are close together they are tightly linked, far apart, almost behave as unlined
Lots of genes are inherited on the same chromosome, a few from crossing over
Recombination frequency depends on the distance between genes
What is a genetic map?
How does recombination frquency enable mapping of genes?
The arrangement and distance between genes on a chromosome deduced from studies of recombination
The frequency of recombanation between two loci gives information with respect to how close the two loci are
1 map unit = 1% recombination
Mapping is more accurate when genes are close together
What is transcription?
Where does it occur?
What are the 3 phases?
What does it need to occur?
The process by which DNA is copied into an RNA message
The nucleus
Initiation, elongation, termination
Needs RNA polymerase, NTPs and a DNA template to occur
Generally what happens in each phase and in processing afterwards?
Initiation: promotor recognition and binding
Elongation: transcription by RNA polymerase II
Termination: sequence dependent termination of RNA growth
Post translational processing from pre mRNA to mature mRNA
What happens in initiation of transcription?
Initiation code is recognised: TATA box, 5’ to 3’
Transcription factors bind here, attracting RNA polymerase
Promoter recognition occurs, the promotor is an area upstream of the ORF which regulates gene expression, includes secquences for binding of transcription factors, RNA polymerase and regulatory factors
RNA polymerase seperates the DNA strands for the RNA nucleotides to bind along the template strand.
What happens in elongation in transcription?
RNA polymerase travels along the template strand picking up base pairs and copying them onto a complementary RNA strand
Reads 3’ to 5’ on template, builds a 5’ to 3’ strand,
identical to coding DNA
What happens in termination of transcription?
End of mRNA production
Dependent on the sequence
What post transcriptional processing occurs to convert pre mRNA to mature mRNA?
CAPPING: 5’ methyl guanine cap. Bonded with 5’ - 5’ triphosphate linkage to stabilise the mRNA. Prevents degradation and has a role in translation.
TAILING: polyadenylation. Stop codon that cleaves mRNA, then 3’ end, lots of adenine nucleotides are added, protection against degradation. Up to 200 As.
SPLICING: premature product of mRNA has introns and exons. Introns which are non coding are removed. Cut by spliceosome proteins which recognise specific sequences. In PKU splicing is wrong
Define
Exons
Introns
Exons are coding portions of DNA
Introns are non coding portions of DNA
What are:
Endonucleases?
Exonucleases?
Endonucleases break within the polynucleotide, non specific or specific
Exonucleases degrade the polynucleotide from the 5’ or 3’ end
What is translation?
Where does it occur?
What does it need to take place?
The process by which the genetic code carried by mRNA is read to produce a sequence of amino acids
Occurs in the cytoplasm
Needs: ribosomes, activated amino acyl tRNAs, mRNA substrates
What are the three stages of translation? Briefly summarise each
Initiation: AUG codon recognition, binding of methionyl tRNA ribosome formation
Elongation: translating, N to C chain growth with addition of amino acyl tRNAs
Termination: stop codon recognition, dissociation of ribosome
Describe initiation in translation
40S subunit with Met-tRNA binds to 5’ cap of tRNA
Anticodon on tRNA binds to AUG
60S subunit then binds
Release of GDP, initiation factors and cap binding proteins
Describe elongation in translation
Met-tRNA occupies the P site
Another aminoacyl-tRNA enters the ribosome to occupy the A site
Needs GTP
Methionine forms a peptide bond with the adjacent amino acid
PEPTIDYL TRANSFERASE: release of water
The tRNA in the P site is now uncharged.
It leaves and the ribosome moves along, translocation
Describe termination in translation
Stop codon is read on the mRNA
UAA, UAG or UGA
No tRNAs that can bind to these codons
Releasing factor pushes out mRNA
Peptide and tRNA are hydrolysed
Protein released into cytoplasm
Carboxy terminal is last
What does a gene contain as well as the information for the protein?
Sequences necessary for expression
promotor
terminator
introns
How is a tRNA activated?
uncharged tRNA
amino acid attaches, activating it
Charged
Created aminoacyl tRNA
Uses ATP
Describe how the amino acids are coded for on the tRNA
tRNA anti codon matches mRNA codon
Inosine base is aspecific
WOBBLE POSITION
Degenerate code
What are the 3 types of RNA?
Explain each
mRNA: messenger
RNA polymerase II, 2%, 100,000s of kinds due to splicing, few copies of each present
tRNA: transfer
RNA polymerase III, 15%, 100 kinds, very many copies of each
rRNA: ribosomal
RNA polymerase I, >80%, few kinds, many copies of each
What is the function of a ribosome?
What kinds are found in eukaryotes and prokaryotes?
How do they read the genetic code?
Used to bind to the mRNA in translation in protein synthesis and provides the location for tRNA
Eukaryotic ribosomes: 80S: 60S and 40S subunits
Prokaryotic ribosomes: 70S: 50S and 30S subunits
Read in triplets with no overlapping and no gaps
How do heterochromatin and euchromatin appear on an electron micrograph?
Heterochromatin: DARK
Euchromatin: LIGHT
Describe the nature of the genetic code
4 letter DNA language
20 letter protein language of amino acids
Triplet code to create 20 amino acids
43 = 64 possibilites
Non overlapping and commaless, no gaps
5’ to 3’ read through
Degenerative, more than one code per amino acid
What are the implications of the degeneracy of the genetic code?
Substitution of bases can lead to a different primary sequence
This can affect the tertiary structure of the protein
Mutation that affects a stop codon can cause it to be longer
Or mutation to create a stop codon: shorter
Some mutations are silent/neutral
Wobble tRNA with iosine
How is gene expression in bacteria different to that in humans?
SImpler promotors
Different transcription factors
One one type of RNA polymerase
Coupled transcription/ translation
No post transcriptional processing
Short lived mRNAs
Simpler ribosomes
Distinctive translation initiation mechanism
Different translation factors
How are differences in human and bacteria gene expression exploited clinically?
Specific examples of antibiotics inhibiting protein synthesis
Bacteria have simpler ribosomes: 30S subunit attacked
Streptomycin inhibits mRNA reading
Tetracycline blocks ribosome A site
Chloramphenicol prevents peptidyl transferase action
Erythromycin prevents translocation of peptidyl tRNA from A to P ste
What are some types of mutation?
POINT: single base change
SILENT: change in base that specifies the same amino acid
MISSENSE: different amino acid coded
NONSENSE: produces a stop codon
INSERTION: addition of one or more bases
DELETION: loss of one or more bases
FRAMESHIFT: not a multiple of 3 bases deleted or added
How can mutations outside of the coding region affect gene expression?
Mutations in promoter regions, where transcription factors bind, can deactivative a gene’s expression or make constantly active
Creation of an alternative splice site, competes with that of normal splice site in RNA processing
Results in proportion of mRNA with improperly spliced intron sequences
Can mutate out a splice site
How can resistance develop in bacteria to anti biotics?
Stops a compound getting in, block entrance of a drug or pump it out
Modify target of a drug: eg a protein
Modify drug that comes in, new enzyme, for example B lactamase changes the B lactam ring of penicillin
Making more of the target, the drug can not cope
What is the constitutive secretory pathway?
What is the regulated secretory pathway?
Constitutive: continuous and unregulated, packaged into vesicles and released by exocytosis.
EG: collagen, serum albumin, immunoglobulins
Regulated: released in response to a signal. Packaged into vesicles but not released until a stimulus is received by a hormone or neurotransmitter.
EG: insulin, glucagon, digestive juices
Describe an overview of the secretory pathway in mammalian cells
- Free ribosomes initiates protein synthesis from mRNA molecule
- Hydrophobic N terminal signal sequence is produced
- Signal sequence of newly formed protein is recognised and bound to by the signal recognition particle SRP
- Protein synthesis stops
- GTP-bound SRP directs the ribosome synthesising the secretory protein to SRP receptors on the cytosolic face of the ER.
- SRP dissociates
- Protein synthesis continues and the newly formed polypeptide is fed into the ER via a pore in the membrane (peptide translocation complex)
- Signal sequence is removed by signal peptidase once the entire protein has been synthesised.
- The ribosome dissociates and is recycled.
What protein modifications occur in the
ER?
Golgi?
ER:
signal cleavage by SIGNAL PEPTIDASE
disulphide bond formation PROTEIN DISULPHIDE ISOMERASE
N linked glycosyltion, oligosacchardies, asparagine side chains with amide group via DOLICHOL
GOLGI:
O linked glycosylation, GLYCOSYL TRANSFERASE, adds sugar to OH group, important in proteoglycans
Timming and modification of N linked oligosaccharides
Further proteolytic processing, exoproteases, endoproteases
Phosphorylation of monnose, to OH group, lysosome signal
What occurs in N linked glycolsylation?
and O linked glycosylation?
N LINKED: in ER
Built up DOLICHOL PHOSPHATE carrier sitting in the membrane
Oligosaccharide transferred to amide group of ASPARAGINE
O LINKED:
-OH groups of SERINE and THREONINE
GLYCOSYL TRANSFERASE, builds up sugar chain from nucleotide sugar substrates
Why is proteolytic procesing important in the formation of some secreted proteins?
ENDOPROTEASES/EXOPROTEASES
N terminal sequence removed in the ER, PRE SEGMENT
Further processing of PRO SEGMENT in the GOLGI
Examples
Preproalbumin
Preproinsulin
How is insulin formed?
PreProInsulin translated, A, B and C peptides
Signal sequence cleaved by SIGNAL PEPTIDASE in the ER
ProInsulin has A, B and C peptides
Endopeptidases cleave the C peptide, in post golgi vesicle, so good measure of endogenous insulin
Insulin: A and B peptides joined by 2 disulphide bridges, made in ER
Active form
Describe the structure of collagen
TROPOCOLLAGEN UNIT
Left handed triple helix of alpha chains
Primary sequence repeats GLYCINE-X-Y
Proline and hydroxyproline usually X and Y
Non compressable, high tensile strength, non extendable
Proline residues maintain correct shape, extended a helix
Hydroxyproline by PROLYLHYDROXYLASE, increase amount of H bonds, needs Vitamin C and Fe2+ for activity
Scurvy, vitamin D deficiency, weak tropocollagen helixes
How is collagen synthesised?
CHASPOGRL
C leavage of signal peptide
H ydroxylation of proline/lysin
A ddition of N linked oligosaccharides to hydroxylysine
S ulphide(di) bond formation
P rocollagen
O linked glycosylation
G olgi to exocytosis
R emoval of N/C terminal peptides
L ateral aggregation to form fibrils
How is tropocollagen cross linked?
Lysine residues to aldehyde derivatives
By LYSYL OXIDASE
Aldehydes then spontaneously form Aldol cross links
Needs vitamin B6 and Cu2+ for activity
Describe how proteins are targeted to the nucleus
Through nuclear pores
Proteins contain a Nuclear Localisation Sequence, NLS
Run of 4 basic amino acids, internal
Folded
Importin recognises NLS, mediates transport inside
Importin binds to Ran GTP, back out
Ran back to nucleus with hydrolysis of GTP
Describe how proteins are targeted to mitochondria
Transported unfolded
Signal sequence, N terminal, amphipathic
Unfolded proteins stabilised by chaperones like MSF (Mitochondrial Import Stimulating Factor)
Signal sequence recognised by TOM (Translocase Outer Membrane)
Those for matrix through TIM (Translocase Inner Membrane)
Needs ATP energy and a membrane potential
Signal cleaved by mitochondrial processing peptidase
Describe how proteins are targeted to the lysosomes
Addition of mannose 6 phosphate to N linked oligosaccharides
N acetyl glucosamine phosphotransferase/phosphoglycosidase
Targeted for M6P addition by signal patch
M6P groups recognised by receptors in the trans Golgi
Vesicles pinched of for transport to lysosomes
Acid pH in lysosymes causes dissociation of the protein and receptor
Receptor back to golgi, phosphate removed from M6P group to ensure it does not travel back to Golgi
What is I cell disease?
Genetic defects in N acetylglucosamine phosphotransferase enzyme
Lack of M6P addition
Lysosomal hydrolases mistargeted for secreted
Found in blood and urine
How are proteins targeted for retention in the ER
Proteins such as disulphide isomerase and signal peptidase, resident in ER
Sometimes lost in vesicles pinched off to go to Golgi
Allows ER proteins to be retrieved
ER residents have a KDEL sequence (lys-asp-glu-leu)
C terinus
Interact wth KDEL receptors in golgi, enhanced by low pH
ER proteins bound to KDEL receptors return to ER in transport vesicles
Dissociate from KDEL receptros in neutral conditions of ER, KDEL back to golgi
Describe the structure and mode of action of selected antibiotics and growth inhibitors
Bacterial cell wall is targeted by PENICLLIN, inhibits TRANSPEPTIDASE ENZYME that forms cross links in the cell wall, osmotic pressure causes cell lysis
Bacterial transcription targeted by RIFAMPCIN, binds to bacterial RNA polymerase preventing transcription
Bacterial protein synthesis targeted by TETRACYCLINE. Competes with tRNA at A site of bacterial ribosome
Anti folates in cancer therapy targeted by METHOTREXATE. Impairs the synthesis of TETRAHYDROFOLATE, which is essential for DNA synthesis, from folic acid. Competitively inhibits DIHYDROFOLATE REDUCTASE (DHFR)
What is cloning?
The product of exact copies of DNA
What is DNA sequencing?
the process of determining the precise order of nucleotides in a DNA molecule
What is restriction analysis?
Describe how it is carried out
Restriction endonucleases (enzymes) are produced by bacteria
They recognise specfic DNA sequences, restrcition sites, and cut double stranded DNA
Restriction sites are often palindromic
Usually produces sticky ends which are staggered cuts
DNA ligase can be used to join the sugar phosphate backbone together
Used with electrophoresis
Investigates the size of the DNA fragements (deletions), investigate mutations, DNA variation (fingerprinting), gene cloning
How are genes cloned using bacteria?
Plasmids used, small circular DNA that can transfer to other bacteria and contains antibiotic genes
Same restriction enzyme used to cut gene of interest and plasmid vector
Hydrogen bonds match sticky ends
DNA ligase joins the backbone
Recombinant DNA created
Introduced into bacterium = TRANSFORMATION
Bacteria with recombinant DNA identified, select for with ampicillin
Bacteria multiply
Explain gel electrophoresis and how it is used to provide information about DNA fragments
Used to seperate DNA fragments of different sizes
Solution of fragments placed in well at negative electrode
DNA molecules are negatively charged - low pK
Travel towards positive electrode
Smaller molecules travel further
Agarose gel used
Buffer used to maintain charge on DNA samples across gel
Power supply generates charge difference
Stain: ethidium bromide fluoresces under UV light, added
What is PCR?
Polymerase Chain Reaction
Amplified DNA segments by repeated copying of target DNA using a thermo stable DNA polymerase and a pair of DNA primers which uniquely define the region to be copied.
What DNA polymerase is used in PCR?
What else does PCR require?
Thermostable
Taq DNA polymerase
from Thermus Aquaticus
Forward and reverse sequence specific primers, short oligonucleotides that can be 3’ extended
DNA nucleotides
Why use PCR?
Amplify specificy DNA fragment
Specific, can discover novel sequences
Template DNA can be minute, crude sample from cheek swab
Investigate single base mutations e.g. Tay Sachs, SCD
Investigate small deletions or insertions e.g. CF
Investigate variation, genetic relationships
Diagnosis of inherited diseases
Detect presence of tumour cells
Early stages of infection
Explain the process of PCR
Denaturation at 95C to spereate strands
Annealing of sequence specific primers to targt DNA, at 55C
Sample is heated to 72C to allow DNA synthesis by Taq polymerise, attaches free nucleotides
Exponential decrease
Extend 3’ end
Describe SDS page
Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis
Seperate proteins on the basis of size only, molecular weight
Detergent, SDS, denatures protein molecules’ secondary and tertiary structure
Gives a negative charge proportional to Mr
One SDS per 2 amino acids
Visualised with Coomassie Blue dye
Why is protein electrophoresis useful?
What does it required?
Needed to check protein levels, enzyme activity etc
Proteins are charged
Will move towards anode or cathode
Can be seperated on the basis of size, shape, charge
Required:
Gel, matrix to allow seperation
Buffer, to maintain protein charge
Power supply, to generate charge difference
Stain, Coomassie Blue
Describe isoelectric focusing
Seperation of basis of charge, isoelectric point
pH gradient across gel
Protein migrates until pI = pH
No net charge at this point so migration stops
Describe 2D page
Can seperate proteins with the same pI
But different molecular weights
Combines SDS PAGE and isoelectric focusing
Gel turned by 90 degrees and run for different property to seperate bands out
Seperate complex mixtures
Important for diagnosing disease states in different tissues, changes in smear intensity/size
Important technique for proteomics, protein analysis
Describe proteomics
Analysis of all proteins expressed from a genome
Digest protein with trypsin
Mass spec
List of peptide sizes
Use database to identify
What is an enzyme assay?
Describe
Measurement of enzyme activity in lab
Clinically useful to tell if an enzyme is present at normal levels
Performed at optimal pH, temperature and ionic strength
Appropriate ions and cofactors must be included
Production of product or disappearance of substrate is measured
Performed at high [substrate] = Vmax
Activity of enzymes in serum often measured, marker of tissue damage
If an enzyme from a particular tissue is found in serum it can indicate tissue damage caused by disease
What are some examples of clinically important serum enzymes and what they mark for
Aspartate transaminase (AST) and alanine transaminase (ALT): markers for liver damage/disease
Creatine Kinase: marker for myocardial infarction
Amylase/lipase: markers for pancreatitis
y glutamyl transferase: marker for liver damage/ alcohol
Alkaline phosphatase: bone disorders
Acid phosphatase (ACP): Prostate cancer
Plasma cholinesterase: Decreased in liver disaease, inhibited in organophosphate poisoning
What is western blotting and what is it used for?
After SDS PAGE
Seperated proteins traansferred to nitrocellulose membrace
Specific proteins can be visualised
Antibodies bind
Which are conjugated to an enzyme or fluorescent label
What is ELISA?
What is it used for?
Describe
Enzyme Linked ImmunoabSorbant Assays
Concentration of a protein can be analysed in a complex mixture by binding of its corresponding antibody
Primary antibody is immobilised on a solid support
Solution to be assayed is applied to the surface
Antibody binds specific protein, others washed off
Second enzyme linked antibody binds to antibody-antigen complex
Binding of second antibody is measured by assaying the enzymes activity, rate at which it converts substrate to colour
What are antibodies?
Bind to specific antigens by recognising a few specific amino acids
Monoclonal: 1 epitope, 1 antigen
Polyclonal: 1 antigen, many epitopes
Epitiope is the part of an antigen recognised by an antibody
What is ELISA used for?
Insulin
Cortisol
Adrenaline
Noradrenaline
TSH
T3/T4
What is Phaeochromocytoma?
Tumour mass secreting lots of adrenaline
What is the sanger chain determination method?
Describe
Dideoxy chain determination
Produced in reads of 300 to 500 bases
Denature template, add labelled primer, DNA polymerase
Add radioactively, fluorescent ddNTPs which lack a OH on C3 as well as on C2
As well as dNTPs
This means no 3’ elongation by DNA polymerase
So the strand terminates
Depending on which ddNTP is used, the new strand will stop at different places
Range of new DNA fragments of different lengths, deantured with heat and seperated by gel electrophoresis
Add to polyacrylamide gel, each runs in a different lane and end result allows us to read the DNA sequence
What is fluorescent dideoxynucleosidetriphosphate sequencing?
All in the same tube
Same lane
Different fluorescent marker for each base
Read by computers, graph
How it DNA currently sequenced?
Fully automated
200 human genomes sequenced per week
Much cheaper
Who would be interested in seeing an individuals genome information?
Ethics
Family
Potential spouses
Doctors
Governments
Police
Schools
Insurance companies
Who owns a genome?
What is the importance of PCR?
Amplifies a small amount of DNA into many many copies, can be a crude smaple
Basis of DNA profiling
What is reverse transcriptase PCR?
RNA is the demplate, instead of DNA
Prior to PCR, a cDNA is made by reverse transcriptase enzyme
Primer is complementary to the poly A tail
RNAse breaks down the RNA afterwards
Introns are left out
What are other reasons for PCR?
(restriction)
Single base mutations can create a restriction site: JUNCTIONAL EPIDERMOLYSIS BULLOSA
Bg/II site created
PCR region around it
cut with Bg/II
Run on gel
Followed by restriction analysis, electrophoresis, DNA sequencing, Southern blotting, another PCR, nested, primers inside
What is hybridisation?
The process of forming a DNA duplex between complimentary nucleic acid strands, normally DNA, for the detection of specific sequences.
Strands are often referred to as the probe and the target
What is a probe?
A stretch of single stranded DNA
Labelled with radioactivity or fluorescence
Used in hybridiation studies
The presence or absence of a complementary stretch of nucleic acid can be established
What is Southern Blotting?
DNA electrophoresis and hydbridisation
Digest with restriction enzymes
Electrophoresis
Transfer from gel to nitrocellulose/nylon, soak up liquid
Hybridised with labelled gene probe to show specific complimentary DNA fragments, in paper bag
Visualised by X ray
Why is southern blotting used?
Investigate gene structure, large deletions or duplications
Investigate gene expansions, triplet repeats, fragile X, Hungtinton’s
Investigate variation, DNA fingerprinting
Investigate mutations in genetic tests, allele specific probes, SCD
What is Northern blotting?
What is Western blotting?
Similar processes
N: RNA
W: proteins
How can allele specific tests be carried out?
PCR: use primers specific for a sequence either side of an allele of interest to amplify. Primers for disease causing mutations. Only amplified if correct mutated primer. 3’ base of primer corresponsd with one allele or the other
Restriction analysis: use one or multiple restriction enzymes with sites in and aruond the allele. Analyse the size of fragments produced. If the restriction enzyme cuts the wild type but not the same, the site is mutated or missing
DNA/Southern Hybridisation: use allele specific oligonucleotides, for wild type and mutant.
What is microarray?
Describe?
ARRAY COMPARITIVE GENOMIC HYBRIDISATION
ARRAY CGH
Screens for sub-microscopic chromosomal deletions for which the locus can not be deduced from the patient’s phenotype
Glass slides with thousands of dots to analyse thousands of genes, minute drops of liquid. Exact position of each gene known. Isolate mRNA, only expressed genes shown. No dot, unexpressed.
- An array of DNA probes covering the entire genome is applied to the surface of a solid matrix
- Patient DNA and normal control DNA are each labelled with different coloured fluorescent TAGs, eg patient red, normal green
- Equal amounts of labelled DNA are then hybridised to the probe array and the hybridisation signals are detected and compared
- For probes where the signal of normal DNA exceeds that of the patients DNA, more green than red, the patient has a deletion of the chromosomal region from which that probe was derived.
Computer compares levels of red and green
Look at deletions and duplications
What tests can be used to analyse DNA at the gene level?
Southern blotting
R T - PCR
Microarray: Array CGH
What is a karyotype?
Picture of a full set of stained METAPHASE chromosomes of an individual, ordered according to chromosome number
Different stains give different banding patterns
Determine sex, chromosome numbers and size
Organised in pairs
Chromosome 1 is the largest
23rd pair is X and Y
What is FISH?
Fluorescent In Situ Hybridisation
Single stranded nucleic acids, usualyl DNA but can be RNA
Permitted to interact so that complexes are formed with sufficiently similar complementary sequences
Investigation of specific DNA sequences on chromosomes inside the cell
Label with fluoresence, can be for specific genes to locate, denature and hybridise
Chromosome painting, each a different colour
Can tell if large chunk are missing
Investigate genes in situ
Investigate chromosome number, structure and behaviour
What is the relationship between changes in nucleotide and amino acid sequences?
Change in genetic code can result in different amino acids being coded for
EG AUA to AUG: Isoleucine to Methionine
A change in the primary sequence of a protein can lead to a change in the shape and therefore function of a protein
What does a mutation =
Sequence variation
What is a point mutation?
A change of a single nucleotide in a nucleic acid sequence
Base substitution
What are the two types of base substitution?
Explain each
Transition:purines to purines, pyrimidines to pyrimidines
Transversion: purine to pyrimidines, pyrimidines to purines, less common
What are the most common type of mutation?
How common are single base substitutions?
Single Nucleotide Polymorphisms: 2/3s of SNPs are C to T
Every 300 bp
How many amino acids are there?
How many codons are there for each?
How many stop codons are there?
20 amino acids
1, 2, 3, 4 or 6 codons per amino acid
3 stop codons
How can mutations in non coding regions or outside genes be detrimental?
Can alter
Binding sites
Promoter sequences
Splice sites
What is the effect of a silent mutation?
One base substitution which does not change the amino acid in which the original codon coded for
Single base substitutions in the 3rd codon position do often not cause an amino acid change
What is the effect of a missense mutation?
Replaces the original specified amino acid with another
Usually a single base change
What codon positions do most non-silent mutations occur at?
1 and 2
What is the effect of a nonsense codon?
Amino acid codon becomes a stop codon
Premature stop codon, PTC
What is the effect of an insertion or deletion of 3 bases?
No reading frame change, but a change in the primary sequence, insertion of amino acid or deletion
What is the effect of an insertion or deletion of 1 or 2 bases
Frameshift mutation
Reading frame of mRNA changed
Shifts triplet code along one or two nucleotides
Often causes a premature stop codon
How can a silent mutation still cause disease?
Can alter splice sites
Might cause a new splice site within an exon
How are PTCs protected against?
Nonsense mediated decay
Degrades PTCs
Little or no of the faulty protein produced
Protection from truncation
What is the meaning of a conservative missense mutation?
Some amino acid substitutions are bettwer tolerated than others
Valine to alanine may be tolerated in non critical regions
Both hydrophobic
Evolved to be next to each other in the genetic table
What is the effect of a mutation in:
Promoter region?
Start/stop codon?
Intron splice sites?
Promoter: transcription factors or RNA polymerase may njot bind
Start/stop: met-tRNA won’t bind, PTC, longer protein
Intron splice: skipping of adjacent exon, if exon 3bp multiple, short mRNA in frame. If not, mRNA short with frameshift, PTC and nonsense mediated decay
How might spontaneous mutations occur?
Not caused by exposure to known mutagen
Errors in DNA replication
DNA bases have slight chemical instability
DNA strand slippage, new strand loops out, extra nucleotide. Template strand loops out, one less nucleotide
Tautomeric shift: proton briefly changes position, altered base properties, behave as different template base for DNA polymerase, C and A, T and G
Rate of spontaneous mutations depends on size and sequence
Usually in non coding regions
How might an induced mutation occur?
Chemicals and radiation
Agents that cause mutations are mutagenic
Agents that cause cancer are carcinogenic
Examples: alkylating agents remove a base
Acridine agents add or remove a base
X rays break chromosomes, delete nucleotides
UV radiation creates thymidine dimers
Nitrous acid, changes base pairing
Ethidium bromide and IQ food mutagen intercalate to force bases apart
EMJ removes purine rings
What is a mutation?
A change in a nucleic acid sequence
Which can be the addition of one or many nucleotides
The removal of one or many nucleotides
The rearrangement of several or many nucleotides
Describe the most common effects of mutations
Not good or bad, just different
Source of genetic variation
Causes a mutant phenotype, differing from the wild type
Mutant allele
What is a wild type?
An individual within a population displaying a wild type trait
The trait which is most common in the population
What type of mutation can be passed onto the offspring?
Occurs in the germline
Gametes
Describe the process of mismatch repair
Enzymes detect nucleotides that don’t base pair in newly replicated DNA
Incorrect base pair is excised and replaced
This is proof reading
Describe the process of excision repair
DNA can accumulate damaged bases through oxidation, alkylation, deamination, uracil
Damaged DNA is removed by excision of bases
Replace a patch of DNA with DNA polymerase
Base excision repair: 1-5 bases, repairs oxidative damage, ROS
Nucleotide excision repair: from external agents, UV damage and carcinogens,, replaces up to 30 bases
What do mutations in the DNA repair protein genes do?
Mutations accumulate
MASH1, 2, 6 genes
Can be inherited
Not efficiently repaired
What is DNA double strand break repair?
When both DNA strands are broken, which can cause chromosomal rearrangements
What is the function of the protein p53?
Monitors the repair of damaged DNA
if damage is too severe = apoptosis
How is cancer caused generally?
DNA damaged to such a huge extent
That apoptosis - promoted by p53 - does not occur
OR
damage leads to uncontrolled growth
So cancerous cells are produced
Describe tumour formation
Derived from individual abnormal cells
Arise from lack of normal growth control
Generated by a multipstep process
More likely to arise from cells undergoing frequent cell division
All of the cells in a tumour are of the same type
Behaviour depends on cell type
What 6 new capabilites do tumour cells have?
Dividing independently of external growth signals
Ignore internal anti growth signals
Avoid apoptosis, p53
Divide indefinitely without aging
Stimulate sustained angiogenesis
Invade tissues to establish secondary tumours
What is an oncogene?
Cancer causing gene
In control of cell division
What is a protooncogene?
key amino acid substitutions can active into dominantly acting cancer causing oncogenes
What is a tumour suppressing gene?
A gene involved in protecting the cell against one step on the path to cancer
How is cancer heritable?
Inherited cancer genes, recessive mutations
Development, dominant inheritance pattern
Initiation of tuimour formation means that both copies are mutated or the functional copy is deleted
Mechanisms of homozygosity: loss of wild type chromosome, deletion of functional gene, point mutation, mitotic recombination
How can viruses cause cancer?
Carry oncogenes
Presence of a virus means that the gene does not function as normal
Why is PCR so important in the diagnosis of genetic disease?
Most mutations are single base changes so are hard to detect
PCR amplifies DNA segments by repeated copying using Taq polymerase, with primers that uniquely define the sequence to be copied
In sickle cell disease the restriction site for MstII is destroyed, so with gel electrophoresis and southern blotting there will be one less DNA fragment digested with MstII
Describe MPCR
Multiplex PCR bases test for most common mutations of a gene
What is MLPA?
Multiplication ligation dependent probe amplification
Exon counts for many exons duplicated in parallel
DNA denatured and mixed with MLPA probes, oligonucleotides containing one of the PCR primer sequences
If both probes hybridise they ligate and are PCRed
Osteogenesis imperfecta and duchenne muscular dystrophy
How can DNA be sampled from an unborn child?
Parent DNA from blood or saliva
Foetal from:
Amniotic fluid cells, 12-20 weeks, guided by ultrasound, culture for 2 weeks, 0.5-1% chance of miscarriage
Chorionic villus biopsy, 10 - 13 weeks, transcervical, abdominal, 2% risk of miscarriage
foetal DNA is motehrs blood isolated
What are some ethical issues associated with genetic testing?
Prantally used in abortion cases
Can screen for disease and prevent and treat harmful effects
Fetal screening, is ti a form of eugenics?
Neonatal can identify abnormalities to treat early
Carrier status, whethere or not to have a child if both carriers
Also children being tested for late onset dominant disease such as Huntington’s, would the older generations want to know?
Insurance access
Employers deciding who would suit jobs
Testing when no cure - psych issues
Selection for better children, messing with God’s creation?
How does EM radiation cause mutations?
Short wavelengths, more damaging
Ionising radiation, produces ions when interacting with cellular moelcules
Solar radiation, X rays, nuclear power plant accidents, environmental sources, food, radon
UV light, all types damage collagen, skin aging
Sun burn and skin cancer from UVB
UV light photons cause adjacent thymine pases to pair, often resolved through spontaneous photo reactivation
What is a chromosome?
How many in a diploid cell?
A nuclear thread like structure which carries the genetic information
Only visible when condensed in cell divisio
23 pairs
Each is one linear DNA molecule
What are chromosomes made of?
CHROMATIN
which is made of:
DNA
Non-histone proteins
RNA
Histones: H1, H2A, H2B, H3, H4
H1 varies between species
H3 and H4 are highly conserved throughout evolution
How is DNA arranged around histones?
Wrapped around an octamer of histones
H2A, H2B, H3 and H4 inside octamer
H1 stabilises
Histones are responsible for the beads on a string structure
Higher order structures are stabilised by hanging loops of DNA onto a protein scaffold, tightly folded chains
What is the difference between euchromatin and heterochromatin?
Euchromatin is lightly packed and under active transcription usually. ACETYL GROUPS, nucleosomes, neads on a string
Heterochromatin is tightly packed chromatin, not being expressed. METHYL GROUPS, solenoids, 30nm fibres
Describe the chromosomal basis of sex determination
Normal Female: 46, XX
Normal Male: 46, XY
What are numerical chromosomal abnormalities ?
A number of chromosomes other than 46
Polyploidy
Aneuploidy
What is polyploidy?
What is a cause of polyploidy?
A number of chromosomes that is a multiple of the haploid number
Common in plants, some animals
Observed in normal human muscle and liver
n=23, 2n=46, 3n=69, 4n=92
Polysperm is a cause, 2 to 3% of all pregnancies are triploid, most miscarry, others die shortly after birth.
Tetraploidy is rarer, usually found at prenatal diagnosis
What is aneuploidy?
An abnormal number which is not a multiple of the haploid number
Monosomy: loss of one chromosome
Trisomy: gain of a chromosome, exists as a triplet
What are the causes of aneuploidy?
Non-disjunction in meitotic cell division, missing or have both homologus chromosomes in one gamete
Non disjunction in mitosis, two populations of cells with different karyotypes, MOSAICISM
Anaphase lag, chromosomes left behind at cell division, because of defects in spindle function of attachment of chromosomes. Laggin chromosome may be lost completely in meiosis or mitosis
What is a structural chromosomal abnormality?
What are the two broad types?
Physical changes to one or more of the chromosomes
Balanced: when the change does not cause any missing or extra genetic information
Unbalanced: when the changes cause missing or extra genetic information
Describe Down Syndrome
47, XY+21
Trisomy 21
Non disjunction
Risk increases with maternal age
Eye problems, hypothyroidism, intellectual disability, congenital heart failure, hearing disorders
Can also arise from chromsomal translocation
What other trisomies are there other than Downs?
Edwards: Trisomy 18, few survive beyound 15 days
Patau: Trisomy 21, multiple congenital abnormalities, most die within a year
What is translocation?
the breakage and reformation of chromosomes such that DNA can be exchanged between chromosomes
Usually results in abnormal phenotypes
Recipricol: between 2 non homologous chromosomes
Breakpoints often lie between genes
Risk of passing one derivative chromosome to offspring, unbalanced, disease
What is Robertsonian translocation?
How does it cause Downs?
P fragments are lost on a copy of CH 21 and Ch 14
This leads to a superchromosome 14:21, as centromeres are acrocentric, near one end
Carrier still has balanced genetic information as p arms are very short
Problesm depend on how chromosome lines up on metaphase plate.
If superchromosome is on opposite side to normal 14 and 21, no problems
If superchromosome is on same side as normal 14, and opposite to normal 21, trisomy 14, and monosomy 21. Both terminate
If superchromosome lines up one same side as normal 21, then trisomy 21 and downs. Other is monosamy 14, not viable
What are interstitial and terminal deletions?
Internally is interstitial
Terminal is at end
By breakage
Always unbalanced
Associated phenotype
May be large enough to see by microscopy
May need FISH
What is duplication?
Some genetic material is doubled within a chromosome
What is an inversion?
No loss of genetic material
Rearrangement within the same chromosome
What is a ring chromosome?
Loss of telomeres or ends of both asrm and formation of a ring
What is an isochromosome?
Creation of two non identical chromosomes
One is a combination of the two short arms
The other is a combination of the two long arms
What happens to inactivated X chromosomes?
They form Barr Bodies at the periphery of the nucleus
Condense
What are some common sex chromosome aneuploidies?
Turners: 45, X. Problems as monosomal for PAR genes in Y chromosome
Triple X: 47, XXX. Learning diabilites, scoliosis, small head, tall
Klinefelters: 47 XXY, male.Reduced testosterone, increased breast tissue, language and reading impairment
XYY syndrome: 47 XYY. Phenotype essentially normal, slightly lower IQ, increased growth rate, normal testosterone
What stain is used in karyotypes?
How is it used for location purposes?
GIEMSA
Banding pattern standardised as a set of ideograms
Dark and light bands are numbered
What cells are used for karyotypes?
Cell culture may be required
Bone marrow 0 to 1 day
Blood 2 to 3 days
Solid tissue, skin
Amniotic fluid
Chorionic villus
7 to 21 days
Which groups of chromosomes have satellites on them?
D: 13, 14, 15
G: 21, 22
What are the types of chromosomes?
How are they grouped?
What are the p arms and the q arms?
Metacentric, acrocentric (D and G), sub metacentric
Grouped according to size similarity and centromere location
P arm is short petit arm
Q arm is long arm
Outline reasons for referall of patients for karyotyping
Congenital abnormalities
Prenatal screening for downs at increased maternal age, family history of chromosomal abnormality, abnormal ultrasound scan
Birth defects, malformations, mental retardation
Abnormal sexual development, eg Kleinfelters
Infertility
Recurrent foetal loss
Acquired: leukaemia and related disorders
What diagnosis offers of chromosomal abnormalities
Accurate diagnosis and prognosis of clinical problems in patients
Better management of affected patients, eg hormone therapy in sex disorders
Understanding of future reproductive risks
Prenatal diagnosis and possible termination of affected pregnancies
