MGD Flashcards
What sort of binding does Myoglobin exhibit?
Hyperbolic
When do you see higher levels of 2,3 BPG and how does it effect Haemoglobin?
Increases in areas of high metabolism + at high altitude
Decreases Haemoglobins affinity for O2
What is the Bohr effect?
H+ and CO2 released during high metabolism bind to Haemoglobin and reduce its affinity for O2
What causes Sickle Cell Anaemia and what happens in it?
Caused by a single nucleotide substitution from Hydrophillic Glutamate -> Hydrophobic Valine
Sticky Hydrophobic pocket allows deoxygenated Hbs to polymerise and distort the shape of the RBC
What causes alpha + Beta Thalassaemia?
Decreased or absent Alpha or Beta chains
Whats the difference between Alpha + Beta Thalassaemia?
Beta - Alpha chains unable to form stable tetramers. After Foetal Haemoglobin goes they have growth problems, anaemia, jaundice, enlarged liver
Alpha - Beta chains can form stable tetramers with higher affinity for O2. Often show no signs or symptoms
What is Vmax?
Maximum rate of reaction when all enzymes active sites are filled with substrate
What is Km?
Substrate concentration that gives half of Vmax
How does the Km value reflect the affinity for the substrate?
The lower the Km the higher the affinity for the substrate
What does CO do?
Binds to Haemoglobin 250x more readily than O2. Increases affinity of other subunits for O2 so they dont release it at tissue
What is Allosteric regulation?
The binding of substrates to one subunit increases the affinity for substrates at other subunits
What is Proteolytic Cleavage?
Enzymes secreted as inactive precursors are cleaved to become active
What causes activation of the Intrinsic blood clotting pathway?
Damaged membrane of RBC’s promotes binding of factor XI to X
What causes activation of the Extrinsic blood clotting pathway?
Trauma releases tissue factor III which activates factor X
What causes Classic Haemophillia?
Defect in factor VIII
What is the name of the fragments made on the lagging strand of DNA when its replicated? Which enzyme joins the fragments together?
Okazaki Fragments
DNA Ligase
Which enzyme causes the unzipping and reforming of DNA during replication?
DNA Helicase
What 4 steps make up the Cell cycle?
G1- Cellular content is replicated
S - DNA is replicated
G2 - Checking phase
M - Mitosis
What are the 6 stages of Mitosis?
Interphase - Replication of DNA
Prophase - Chromosomes condense, Nuclear membrane breaks down, Spindle fibers form
Metaphase - Centrioles move to poles, Chromosomes line up along center
Anaphase - Chromosomes pulled apart
Telophase - Nuclear Membrane reforms
Cytokinesis - Cytoplasm divides
What is the difference between Genotype + Phenotype?
Genotype - Genetic make up of an individual
Phenotype - All observable characteristics of an individual as a result of their genetic make up
How is RNA synthesised in Transcription?
Initiation - Initiation code (TATA) is recognised, Transcription factors bind here (promoter region). RNA Polymerase is atracted
Elongation - RNA Polymerase travels along template strand from 3’->5’ picking up free nucleotides
Termination - Addition of Mythyl - Guannine cap to 5’ end to stabalise RNA. At 3’ end there is stop codon followed by a Poly A tail
Splicing - Introns removed by endonucleases
What is the process of Translation in DNA replication?
Initiation - 40s Subunit binds to 5’ end of mRNA, start codon is 5’AUG. 60s Subunit then binds
Elongation - rRNA has P-site for holding peptide chain + A-site for accepting new tRNA. tRNA enters sA-site, forms peptide bind with tRNA in P-site, tRNA in P-site is then uncharged so leaves P-site
Termination - Requires a stop codon, no tRNA can bind to these codons
What does it mean that the triplet code of DNA is degenerate? Why is this beneficial?
There is more than one code per amino acid
Means that not every mutation changes the primary sequence of DNA
How is the active form of Insulin formed?
Synthesised as Preproinsulin,this contains signal sequence, C peptide, A+B peptides.
Signal sequence is removed from N-terminal by signal peptidase
3 disulfide bonds form - Proinsulin
Molecule cleaved into 3 peptides, C peptide released
A+B held together by 2 peptide bonds - Active Insulin
What can be used to measure endogenous Insulin levels in diabetics?
C peptide released from Insulin production
How are proteins targeted to the ER?
Signal sequence on N-terminal is recognised by Signal Recognition Protein + cleaved by Signal Peptidase
Protein is unfolded during transfer into the ER
How are proteins targeted to the Nucleus?
Nuclear Localising Signal is found on the surface of the protein
Importin recognises NLS and mediates transport + transfer
Signal is retained so proteins can be reimported during nuclear reformation after cell division
How are proteins targeted to Mitochondria?
Amphipathic signal for initial targeting to matrix (found on N-terminal) is cleaved
Protein is held partially unfolded by Mitochondria Import Stimulating Factor, TOM + TIM form the import channel
How are proteins targeted to Lysosomes?
Mannose-6-Phosphate signal is added to N-linked Oligosaccharides in Golgi
M6P receptor of Golgi sends protein to lysosome, enters folded in vesicle
Vesicle + receptor return to Golgi
How are proteins retained within the ER?
ER resident proteins have KDEL at C-terminus, if transported to Golgi KDEL receptors bind due to low pH and transport protein back to ER
Receptor dissociates from signal due to neutral pH and returns to Golgi
How does Penicillin work?
Targets Bacterial cell wall by inhibiting Transpeptidase enzyme that forms cross links in cell wall
How does Rifampicin work and what is it used to treat?
Targets bacterial RNA Polymerase, prevents mRNA synthesis so no translation + bacteria can’t divide
Used to tread TB, Leprosy, Staphylococcus, Meningitis
How does Tetracyclin work and what is it used to treat?
Targets bacterial ribosomes, Binds to 30s subunit blocking the A site so tRNA can’t bind
Used to treat UTI, Gonorrhoea, Chlamydia, Pneumonia + eye infections
In what ways can Antibiotic resistance occur?
- Decreased influx of drugs by expressing a reduced number of carrier proteins
- Increased Efflux of toxic products from the cep (Multi-drug resistance protein I)
- Increased transcription of target to overwhelm the drug
- Specific target of drug acquires a mutation lowering the affinity for it
How does DNA sequencing by Sanger Dideoxy Chain Termination Method work?
Add fluorescent ddNTP’s (Dideoxynucleodie triphosphate) are added with regular dNTP’s to a DNA strand with DNA polymerase. ddNTP lacks a 3’ OH so terminates strand. Different strand lengths used to determine position of different nucleotides
How does Restriction Analysis work?
Restriction endonucleases are bacterial enzymes that recognise specific sequences and cut them, this produces fragments that can then be analysed by gel electrophoresis
How is Southern Blotting performed?
Unlabelled DNA from electrophoresis is transferred with nylon. DNA heated to 95 degrees to allow specific gene probes to bind. This can then be used to investigate gene structure
How does Western Blotting work?
Primary antibody is added and binds to protein of interest. Secondary antibody is conjugated with a label and binds to Primary antibody
What does SDS PAGE do and how does it work?
Proteins are separated by molecular weight. Detergent is used to break the tertiary structure and gives them a charge relative to their molecular weight. Smaller molecules will run further than larger ones
What does Isoelectric Focusing do?
Proteins are separated based on their isoelectric points.
Stable pH gradient is established in gel and proteins will migrate until it reaches a pH that matches its pI
What does 2D-PAGE do?
Seperates proteins by isoelectric point in one direction and by molecular weight in the other
What is an Enzyme Assay used for? How is it done?
Used to measure the activity of an enzyme.
Performed at optimal pH, temperature and ionic strength, the production of product or disappearance of substrate is constantly monitored
What are the two main markers for Liver damage?
Aspartate Transaminase
Alanine Transaminase
What are the three main markers for an MI?
Creatine Kinase
Lactate Dehydrogenase
Cardiac Troponin
What is the main marker for Pancreatitis?
Amylase
How does Enzyme-Linked Immunoabsorbent Assay (ELISA) work? Whats it used for?
Primary antibody (specific to protein) is immobilised on solid surface.
Solution to be assayed is applied to antibody coated wall.
Secondary antibody conjugated with an enzyme binds to the antigen-antibody complex. Substrate is then added and converted by enzyme into coloured product.
Binding of secondary antigen is proportional to concentration of protein.
Used to determine concentration of a protein
How does Reverse transcriptase - PCR work?
RNA template is converted into cDNA using a reverse transcriptase and the mRNA strand is digested and the cDNA is used as a template strand for PCR to take place
What is Karyotyping?
Cells are arrested during cell division and chromosomes are stained. Chromosomes are ordered by length under microscope
How does Fluorescence in Situ Hybridisation work?
Probe DNA is labelled with fluorescent dye, DNA is denatured and allowed to hybridise. Shows whether specific DNA is present and where its located
What are the two types of Chromatin?
Euchromatin - Lightly packed chromatin under transcription
Heterochromatin - Tightly packed chromatin, genes not expressed
What is Polyploidy? What commonly causes it?
When an embryo gains an extra set of chromosomes (a haploid set of chromosomes)
Commonly caused by polyspermy
What is Aneuploidy? How does it occur?
Abnormal number of chromosomes that is not a multiple of the haploid number
Occurs because of the failure of a chromosome to separate properly
Monosomy is the loss of a chromosome
Trisomy is the gain of a chromosome
