MCBG Flashcards
What is the difference between heterochromatin and euchromatin?
Heterochromatin is tightly packed solenoid of DNA that appears dark in an electron micrograph. The genes here are not expressed.
Euchromatin is DNA which resembles ‘beads on a string’ and appears lighter in an electron micrographs. The genes here are expressed.
Which organelle is the largest in the cell?
Nucleus
What appears as a dark dot in the nucleus and what is its function?
Nucleolus-repeated sequences that have the genes required for ribosome production found on 5 chromosomes that aggregate as heterochromatin appearing as a dark circle in the nucleus. Consists of ribosomal RNA and proteins as it is the site of ribosome production.
What are the functions of the nucleus?
DNA synthesis and repair
RNA synthesis
Ribosome assembly
Describe the fluid mosaic model of biological membrane structure.
Amphipathic phospholipids -hydrophobic tails (fatty acids) pointing inwards + hydrophilic heads (glycerol+choline+phosphate) pointing outwards, bilayer impermeable to water and water soluble substances, permeable to lipid soluble substances
Proteins embedded in various ways- alpha helix of proteins is hydrophobic so anchors protein to membrane.
Proteins can be attached to other proteins in the membrane or attached to non-protein molecules in the membrane. Allows selective permeability.
What are the functions of the plasma membrane (plasmalemma)? (6)
Selective permeability (which maintains electrical gradient), intercellular recognition, signal transduction, intercellular junctions (lateral domain), endocytosis, exocytosis, transport of materials across surface from apical domain
What is the glycocalyx?
Cell coat made up of polysaccharide side chains on the outside of the plasma membrane. This is because many proteins embedded in the phospholipid membrane are glycocylated and this specific structure which allows specific recognition.
What are the functions of RER?
- synthesised phospholipid (membrane)
- ribosomes on surface synthesis proteins and proteins are transported into cisternae where they can be glycocylated
- proteins transported to golgi apparatus in vesicles
Where are SER abundantly found?
Cells involved in lipid production (eg. Liver, mammary gland)
Cells involved in steroid production (eg. Ovary, testis, adrenal gland)
What is the main function of SER in muscle cells?
Store and release of Ca2+
What are the functions of SER? (2)
Lipid (mammary glands) and steroid synthesis (ovaries/testes)
What is the function of the golgi apparatus?
Modifies, sorts, concentrates and packages proteins (cis to trans) formed on the RER for secretion from the cell, inclusion in lysosomes and incorporation into the plasma membrane.
Where are phospholipids and hence membranes formed?
RER
Where are lysosomes produced?
Golgi apparatus
What is the function of lysosomes?
Consist of hydrolytic enzymes to allow intracellular digestion.
What is the difference between the enzymes in lysosomes and peroxisomes?
Lysosomes consist of hydrolytic enzymes whereas peroxisomes consist of oxidases and catalases.
Where are peroxisomes abundantly found?
Liver and kidney cells
In which cells are mitochondria found?
What colour do cells with abundant mitochondria appear?
All cells apart from red blood cells
Brown-mitochondria have a slight brown tinge
Why can mitochondria divide unlike other organelles? How do they divide?
They have their own circular DNA and ribosomes.
Divide by binary fission.
What are microfilaments in cells?
Actin filaments involved in muscle contraction, intracellular movement (microvilli) and help form the cytoskeleton.
What is the purpose of intermediate filaments in cells?
Resist mechanical forces to the cell. They found a tough supporting mesh work in the cytoplasm and beneath the nuclear membrane forming the nuclear lamina. They attach to desmosomes. Found in epithelia.
What are the functions of microtubules?
Support the cell by giving its shape, involved in intracellular and cellular movements (cilia and flagella if present).
What is gives the shape of microvilli?
Actin microfilaments
What is present in cilia?
Microtubules in a 9+2 structure.
9 doublets around circumference
2 singlets in the centre
How is chromatin activated?
Inactive chromatin is methylated and deacetylated. Demethylation and acetylation activates the chromatin.
What is the difference between a nucleoside and a nucleotide?
Nucleosides have a sugar, a base but do not have a phosphate group.
Nucleotides have a sugar, a base and a phosphate group.
What is the difference between ribose and deoxyribose?
On carbon 2 of the pentose sugar, there is a hydroxyl group. On carbon 2 of 2-deoxyribose sugar, there is no oxygen and just a hydrogen.
On which carbon is the phosphate attached in DNA and RNA?
Carbon 5
On which carbon is the base attached in DNA and RNA monomers?
Carbon 1
Why is DNA negatively charged?
Phosphate groups are negatively charged.
Which bases are purines and which are pyrimadines?
What are the differences between them?
Purines-adenine, guanine (2 carbon rings)
Pyrimadine- thymine, uracil, cytosine (1 carbon ring)
How do hydrogen bonds vary between the bases?
Adenine to thymine/uracil (2 H bonds)
Guanine to cytosine (3 H bonds)
Proteins involved in the regulation of DNA transcription bind to DNA. Do they bind to the minor or major groove of the double helix and why?
The major groove because this is where the sugar phosphate backbone is furthest apart so the proteins can interact with the bases (no steric hinderance). Proteins bind to a specific sequence so the bases must be protruding.
What reaction occurs to join DNA/RNA molecules?
Pyrophosphate hydrolysis
Formulate an equation for DNA replication.
(dNMP)n + dNTP —> (dNMP)n+1 +PPi
How does termination occur in DNA replication?
The replication forks meet
How do Okazaki fragments combine?
DNA ligase
How do the DNA strands separate during DNA replication?
DNA helicase
What 3 steps are involved in DNA replication.
Initiation- DNA helicase seperates the strands of DNA forming replication forks. DNA primase binds to the 3’ ends of DNA strands.
Elongation- DNA polymerase extends the chain by continuous or discontinuous replication
Termination-DNA ligase combines the okazaki fragments. Replication forks meet.
What are the substrates for DNA replication?
Deoxyribose nucleotide monophosphates or Deoxyribose nucleotide triophosphates?
dNTP’s
The ends of DNA molecules in chromosomes are continually shortened as replication goes on. How are genes not lost?
There are telomeres at the end of the chromosomes which are repeated DNA sequences that do not code for proteins.
Why are nucleoside analogues used instead of nucleotide analogues in anti-cancer and anti-viral drugs?
Nucleosides do not have a phosphate group whereas nucleotides have a phosphate group.
Nucleosides can pass through the cell surface membrane and nuclear membrane easily whereas nucleotides are much larger and cannot.
When nucleosides are incorporated into the DNA chain, DNA polymerase cannot further extend the molecule, inhibiting DNA replication.
What type of error in DNA replication can lead to addition of a new nucleotide or deletion of a nucleotides?
Slippage-usually occurs with microsatellites (trinucelotide repeats eg. CAG in HTT gene).
The template strand can ‘loop out’ resulting in the newly synthesised strand having deletions.
The newly synthesised strand can ‘loop out’ resulting in the newly synthesised strand having expansions.
All of the DNA molecules synthesised using this strand as a template will possess the mutation.
What disease is caused by multiple additional repeats of a micro satellite? What DNA error mechanism causes this?
Huntington’s disease
Slippage
What type of DNA replication error causes double strand breaks to form from single strand breaks? A mutation in what gene causes the double strand breaks to persist?
Defects in response to fork blockage.
BRCA gene repairs fork. Mutation in BRCA gene means the fork is blocked so when replication continues, the single strand breaks form double strand breaks. Chromosome integrity is lost and macro-mutations can occur.
What is Werner syndrome?
Werner syndrome is a genetic disorder which is recessive. Werner protein is a helicase which is important in DNA replication. In Werner syndrome, there is a mutation in the gene that codes for this protein causing DNA replication defects. Patients exhibit premature ageing.
Why is DNA packaging important?
To fit 2m of DNA in a 6 micrometre nucleus
What is a gene?
A gene is a specific sequence of DNA at a specific loci on a chromosome that codes for the production of protein(s).
If the wrong nucleotide is incorporated onto a DNA strand, what about the nucleotide is important for the strand to be extended?
When you are adding on a nucleotide into a DNA strand, it is in 5’ to 3’ direction. Therefore, the 3’ end needs to bind to the next nucleotide for it to be extended. If there is no hydroxyl group on the 3’ end for a phosphate from another nucleotide to bind to, elongation of the chain will stop.
What is the difference between exonucleases and endonucleases?
Exonucleases cut nucleotides from the end of DNA molecules. They are usually 3’ or 5’ specific.
Endonucleases cut nucleotides from within the DNA molecule.
What are the functions of DNA polymerase?
Catalyses the formation of phosphodiester bonds between dNTP’s
Proof-reading- 3’ to 5’ exonuclease ability enables it to remove incorrect nucleotides which it has incorporated and insert the correct one.
A typical human cell divides once every 22 hours (G1=7.5h, S=8h, G2=5h, M=1.5h). Every human diploid cell contains approx 6000million bp of DNA. The speed of a DNA replication fork is about 100 nucleotides per second. What is the minimum no. Of origins of replication per chromosome?
23
What is nondisjunction?
Failure of homologous chromosomes or sister chromatids from separating normally during nuclear division usually resulting in abnormal chromosomal distribution in daughter nuclei (aneuploidy)
If you are told that in a cell division of human cells 23 unduplicated structures are seen to
travel to each pole, can you tell which stage of meiosis or mitosis this is?
Meiosis II, anaphase II
What causes genetic diversity during meiosis?
Random assortment of homologous chromosomes
Crossing offer between sister chromatids
What is G0 and why does it exist?
Cells that don’t divide go through g0. Can be temporary or permanent. This occurs when there are no signals for the cell to divide. Static cells- cardiac muscle, skeletal muscle
How do homologous chromosomes pair up during prophase I of meiosis? How do the sex chromosomes do this?
Homologous chromosomes have the same genes along their length so identical sequences enables them to recognise one another. The sex chromosomes have some common genes along their length.
What is the main aim of many cancer treatment?
DNA damage- affects healthy cells.
How do pre-malignant cells become malignant?
DNA damage response is switched off.
What is the difference in the percentage of abnormal offspring when nondisjunction occurs in meiosis I and meioisis II?
Meiosis I - 100%
Meiosis II - 50%
What is mosaicism?
The presence of one or more cell lines in an individual.
What causes and affects the extent of mosaicism?
When nondisjunction occurs- meiosis I, meiosis II, mitosis
If a mitotic division- whether it occurs in the first zygotic division or after
During the lifetime of a female, how does the meiotic stage are her gametes change?
In a foetus, primary oocyte arrests in prophase I. During ovulation, secondary oocyte arrests in prophase II. Full meiosis only occurs at fertilisation.
How can interfering with telomerase be a treatment for cancer?
By inhibiting telomerase in cancer cells, telomeres are no longer added to the ends of chromosomes so as replication continues, genes will be lost leading to cell death.
How does UV light damage DNA?
Creating pyrimadine dimers. These are bonds between adjacent thymine and cytosine bases.
Person 1 has a disease in which the gene for nucleotide excision repair is mutated. Person 2 is healthy. Person 1 and person 2’s DNA is radiated with UV light, denatured and ultra centrifuged. Person 1 showed no reduction in weight whereas Person 2 showed significant reduction in weight of DNA, before and after being radiated by UV light. Why?
In healthy individuals, the damaged DNA would be removed by nucleotide excision repair mechanisms. When this nucleotide excision repair occurs, short sections of single stranded DNA are removed making shorter strands of DNA. Whereas in XPG patients, the damaged DNA are not removed by nucleotide excision repair because the enzyme required for this is not formed. Therefore, the DNA in a healthy individual would be shorter.
How is spindle polarity regulated?
A bipolar spindle has 2 centrosomes. One centrosome is 2 centrioles.
What is cohesin?
Molecular glue that holds sister chromatids together in a replicated chromosome
Histones have an isoelectric point of 10.8. What charge do histones have under physiological conditions? What amino acids must be present to achieve this?
Positive charge
Basic hydrophilic polar
The side chain of aspartic acid has a pKa value of 2.8. What does this mean?
At a pH of 2.8, there is exactly the same concentration of protonated and deprotonated forms. It has a low pKa which shows it is acidic.
Explain why some amino acid side chains are charged at physiological pH.
Some amino acids are acidic or basic so they are protonated (positively charged) or deprotonated (negatively charged) at physiological pH.
If the pH is lower than the Ka of an amino acid, are there more deprotonated or protonated species of the amino acid?
There is a higher concentration of hydrogen ions than there are at the half equivalence point so equillibrium shifts to the left and there are more protonated species of the amino acid.
The isoelectric point of a protein is 9. What kinds of amino acid residues are responsible for this property?
A low concentration of hydrogen ions is required to neutralise the protein so the protein has many positive, protonated amino acid residues. These are positive, polar and hydrophilic.
Which amino acids are strong helix formers?
Alanine and leucine
Which amino acids are helix breakers?
Glycine and proline. These are small hydrophobic residues.
Where do hydrogen bonds form in an alpha helix and in a b pleated sheet? Which bonds are important for this to occur?
There are hydrogen bonds between the H of N-H and O of C=O. Rotation about the C-N and C-C bonds are important in determining the secondary structure.
In an alpha helix, this is between every 4 amino acid residues.
In a B pleated sheet, this is between 2 anti-parallel or parallel B strands.
What allows hydrogen bonds to form the way that they do in an alpha helix?
Rotation about the bonds next to the peptide bonds to create specific bond angles (C-N and C-C)
Suggest why proline and glycine are helix breakers?
Glycine has a very small R chain which supports other more compact structures.
Proline has a large R group that does not allow rotation about the C-N bond to create the specific bond angle required to form an alpha helix
What are motifs and domains in proteins?
Motifs are folding patterns containing 1 or more elements of secondary structure.
Domains are part of a polypeptide chin that folds into a distinct shape and these often have a functional role.
What are the properties of peptide bonds (3)
Planar
Rigid-resonance of C=O bond means it has double bond properties
Trans-the alpha carbons are on opposite sides of the bond due to steric hinderance in the cis form
What bonds are present in the secondary structure of a protein?
Hydrogen bonds
What bonds are present in the tertiary and quaternary structure of a protein?
Disulphides bonds, ionic bonds, hydrophobic forces, van der vaals forces and hydrogen bonds between R groups,
How are hydrophobic forces formed between amino acid residues in a polypeptide?
Amino acid residues with hydrophobic side chains cluster together to displace water.
How are electrostatic forces formed between polypeptide chains?
Positively charged and negatively charged amino acid residues interact with one another to form salt bridges.
How can disulphides bonds be broken? Between which residues are they found?
Between cysteine residues
Can be broken with reducing agents
Which amino acids are acidic? What bonds can they form?
Aspartic acid and glutamic acid, hydrogen bonds and ionic bonds when negatively charged
Which amino acids are basic?
Histidine lysine and arganine
How does altering pH disrupt protein structure?
Changes ionic and hydrogen bonds by altering the ionisation states of the amino acid residues.
What are amyloid fibres?
A misfolded insoluble form of a protein which is normally soluble. This is caused by a core B pleated sheet forming before the rest of the protein. The links between the B chains are strengthened by hydrophobic interactions between non-polar hydrophobic aromatic residues.
What is the transition state in an enzyme catalysed reaction?
A high energy intermediate that lies between substrate and product
What is activation energy?
Activation energy is the minimum energy required by a substrate for a reaction to occur
How do enzymes work?
Enzymes are biological catalysts that increase the rate of reaction by providing alternative pathways of lower activation energy. They facilitate the formation of the transition state (a high energy intermediate between substrate and product)
Are active sites formed by amino acid residues adjacent to one another in the primary sequence or amino acid residues from different parts of the primary sequence?
Amino acid residues from different parts of the amino acid
Does the active site form a complementary shape to the substrate before or after binding with the substrate?
After binding with the substrate
How are substrates bound to the active site?
Weak bonds-bonds that are not covalent
2 enzymes have the following Km values.
Enzyme 1= 53 micromoles
Enzyme 2= 0.053millimoles
Which has the highest affinity for its substrate?
Neither, equal affinities
2 enzymes have the following Km values.
Enzyme 1= 50micromoles
Enzyme 2= 0.075millimoles
Which has the highest affinity for its substrate?
Enzyme 1 because it has a lower Km value.
A lower concentration of substrate is required to reach half of Vmax
What is Vmax?
The velocity of a reaction when maximum enzyme substrate complexes have been formed
What does the Michaelis-Menten model predict?
A specific complex between the enzyme and substrate is a necessary intermediate in catalysis predicting a rectangular hyperbola of reaction velocity against substrate concentration.
Hexokinase is always active whereas Glucokinase only becomes active when glucose levels peak after feeding. Which one requires a lower Km value and why?
Hexokinase because it needs a higher affinity for the substrate so it can bind to substrate when there are low concentrations of glucose present.
What is a unit?
1 micromole of product formed per minute
The activity of enzyme in 1ml of serum was measured to be 7.7nmol per minute. What is the activity of the enzyme in units per L.
7.7 micromoles per L
Is Km or Vmax affected in competitive inhibition?
Km
Is Km or Vmax affected in noncompetitive inhibition?
Vmax
How can you easily estimate Vmax and Km?
Using a Lineweaver-Burk plot
Competitive and non-competitive inhibition are both types of….. inhibition
Reversible
Reversible inhibition involves which type of bonds?
Bonds that are not covalent and can freely dissociate
Which alleles are dominant and recessive in the ABO blood group system?
IA is dominant over IO
IB is dominant over IO
IA is codominant over IB
By looking at a pedigree, how can you tell if a condition is inherited in an autosomal recessive manner?
It skips generations
By looking at a pedigree and the condition is more common in males than females, in what manner is it inherited?
X-linked recessive
How can you tell whether something is inherited in an X-linked dominant manner or autosomal dominant manner?
Males can only pass it on to their daughters
What are linked genes? What is an important characteristic of this genes in meiosis?
Genes on the same chromosome. The closer together they are, the more tightly linked they are. They do not show independent assortment during metaphase of meiosis I so they are usually inherited together.
When are you likely to have a high recombination frequency? What is the maximum recombination frequency? Why?
The further apart genes are because the chiasma can occur at multiple points between them.
0.5 - at this point there is an equal number of even and odd recombinations
Does a gene include the signalling sequences involved in transcription regulation?
Yes
What is the most abundant type of RNA?
rRNA
Where does DNA transcription occur?
Nucleoplasm
Describe the initiation process of DNA transcription?
Promoter sequence including TATAAA recognised
Transcription factors bind
RNA polymerase II binds here
What sets the direction for transcription
TATAAA on the coding strand
——->
How can DNA transcription be increased?
There may be enhancer sequences upstream to the promoter sequence to which activators bind. When activators bind, mediators are released which binds to the transcription factors at the promoter sequence, increasing the rate of transcription
What reaction occurs to form the phosphodiester bonds between rNTP’s during transcription?
Pyrophosphate hydrolysis
Describe how DNA transcription terminates?
A specific sequence on the mRNA is recognised and cleaved by an endonuclease. The mRNA that had been replicated downstream to this degrades and RNA polymerase II falls off.
What prevents degradation in pre-mRNA?
A 5’ guanine cap which forms a 5’ to 5’ link with the pre-mRNA
A 3’ polyA tail consisting of up to 200 A’s
How is translation initiated?
The 5’ guanine cap is recognised by the methionyl tRNA so the mRNA enters the 40s subunit of the ribosome. The 60s subunit of the ribosome binds then binds to methionine acyl tRNA. The AUG codon enters P site and the second codon on the mRNA enters the A site so the second tRNA is recruited.
What enzyme is involved in the formation of peptide bonds between amino acids on adjacent tRNA molecules? What is its function?
Peptidyl transferase
Breaks the bond between tRNA and its amino acid.
When does termination of translation occur?
When a stop codon enters the A site. UAA UGA UAG.
How does termination of translation occur?
Protein release factor binds to the stop codon causing the mRNA to be released from the ribosome. Addition of water to the polypeptide removes it from the tRNA in the P site.
At which site does the tRNA enter in a ribosome?
A site
What is the difference between prokaryote and eukaryote ribosomes?
Prokaryote- 70s —->30s and 50s
Eukaryote- 80s —-> 40s and 60s
What is the start codon on mRNA and what amino acid does this code for?
AUG methionine
What is a wobble base?
The last amino acid in the anticodon of tRNA read 3’ to 5’ is wobbly. This means one type of tRNA can recognise more than one codon. This is because this base can be:
Inocine - binds to A C U
U - under some circumstances binds to G
Why is the genetic code degenerate?
There is more than one codon for some amino acids. There are 64 possible codons for 20 amino acids
What is a charged tRNA called?
Where on the tRNA does the amino acid bind?
What is the structure of tRNA?
Amino acyl tRNA
Amino acid binds to 3’ -OH
Single RNA polypeptide, folds on itself to form hydrogen bonds between the antiparallel backbone in a clover shape.
In which direction do proteins grow?
N terminal to C terminal
What types of modifications occur to proteins after translation?
Proteolytic cleavage
Chemical modification
What type of modifications to proteins occur in the ER? (5)
What are the proteins that enter the ER destined for?
Proteolytic cleavage
N-linked glycosylation
Formation of disulphide bonds between cysteine residues
Correcting of misfolding by chaperone proteins
Assembly of multi sub-unit proteins
Hydroxylation of selected Lys and Pro residues
Proteins destined to be: Secreted into the extracellular environment Embedded in the membrane Stay in the ER Transported to lysosomes
What types of modifications to proteins occur in the golgi? (3)
O-linked glycosylation- important in the formation of proteoglycans (ECM in CT)
Removal of mannose
Sulfation of tyrosine and carbohydrates
Why do modifications to proteins occur after translation?
All proteins have a unique 3-D structure but some proteins require further modification to become active after translation.
What are the requirements for protein sorting?
- A signal intrinsic to the protein
- A receptor that recognises the signal and directs it to the appropriate membrane
- A translocation machinery
- Energy to transfer the protein to the new place
What are the possible destinations for a protein that has been synthesised by free ribosomes?
- remain in cytosol
- imported into an organelle eg. Peroxisome, mitochondria, nucleus
Where are proteins destined for the secretory pathway synthesised?
On the ER membrane
Where are polypeptides that are synthesised on the ER destined for?
The plasma membrane of the cell
Extracellular environment
To remain in the ER
Lysosomes/endosomes
What are the different types of secretion?
Constitutive and regulatory
Give 2 examples of constitutive proteins.
Collagen and albumin
Give three examples of regulatory proteins.
Endocrine secretions-Hormones eg. Insulin
Neurocrine secretions-Neurotransmitters
Exocrine secretions-digestive juices
What sequence is present on all proteins destined for the secretory pathway?
The signal leader amino acid sequence at the N terminal of the protein. 5-30 amino acids long and forms an alpha helix. The central component is rich in hydrophobic residues so it can pass the membrane easily.
How do membrane proteins become embedded into the membrane of the ER?
A stop transfer sequence halts transfer of the peptide across the membrane
What sequence is present on a protein destined to remain on the ER?
There is a KDEL sequence present on the C-terminus.
Why does transport of proteins into the ER not require energy?
The pH in the ER is about 7 whereas the pH in the golgi is about 6.5 . The affinity of the KDEL receptor for the KDEL sequence is higher in the golgi and lower in the ER. The difference in pH facilitates the movement of the protein.
What is the difference between the signal sequence on proteins destined for lysosomes and other signal sequences?
The signal sequence for proteins destined for lysosomes is mannose-6-phosphate present in the N-terminus of the protein. This is formed by N-linked glycocylation in the golgi when a sequence on the protein is recognised.
All other signal sequences are sequences of amino acids within the protein.
Which signal sequences are not cleaved?
KDEL sequence (proteins destined for ER) and NLS sequence (proteins destined for nucleus)
Proteins destined to which compartments remain unfolded? How?
Proteins undergoing co-translational insertion into ER. Translation is still occurring.
Mitochondria. Chaperone proteins keep the protein unfolded. This requires energy.
Explain the post-translational processing of insulin.
Preproinsulin - Signal leader sequence on N-terminal directs it to be co-translationally inserted into the ER membrane. Signal sequence is cleaved by signal peptidase
Proinsulin- disulphide bonds between cysteine residues in ER, endopeptidase cleaves part of the polypeptide in golgi and O-linked glycosylation occurs
Insulin- Packaged in a vesicle and released from cell by exocytosis
Which is the most abundant protein in the body?
Collagen
What is type 1 collagen made up of? List some sites where this is found.
Two alpha 1 chains and one alpha 2 chain in the tropocollagen.
Bones, skin, tendons, ligaments
Which cell secretes collagen?
Fibroblasts
What is the first step of collagen biosynthesis.
Pre pro alpha chains are directed to the ER for co-translational insertion into the ER due to the signal leader sequence on the N terminal rich in hydrophobic residues.
What occurs to the pro alpha chain in order to form pro-collagen? (5)
The proline and lysine are hydroxylated in the ER by prolyl hydroxylase and lysyl hydroxylase so hydrogen bonds form between the alpha chains which holds together the triple helix
There is N-linked glycosylation in the ER. Galactose is added in the ER
Disulphide bonds form links between the N-terminals and C-terminals of the alpha chains
Triple helix forms. Transported in vesicle to golgi.
Further o-linked glycocylation in the golgi.
What is the difference between pro-collagen and tropocollagen?
Pro-collagen becomes tropocollagen when released from the cell by exocytosis as there are peptidases present in the extracellular environment that cleave the N-terminal and C-terminals of the alpha chains so only the triple helix is left.
How does tropocollagen form collagen fibrils?
Tropocollagen aggregates laterally to form rows of tropocollagen with gaps in between. They are arranged randomly. There are cross-links between the tropocollagen molecules formed by Lysyl oxidase.
What gives collagen fibres a stripy appearance in an electron micrograph?
In the collagen fibrils, there are gaps between the tropocollagen. Metals deposit here appearing dark. Where the tropocollagen lies, it appears lighter.
Describe the structure of tropocollagen.
Made up of 3 alpha chains forming a triple right handed helix held together by hydrogen bonds
The alpha chains have Glycine- x - y repeats. Where x and y is usually lysine, proline or hydroxylysine and hydroxyproline
What are the properties of tropocollagen?
High tensile strength
Non-extensible
Non-compressible
List the different structures involved in the biosynthesis of collagen.
Prepro alpha chain Pro alpha chain Pro collagen Tropocollagen Collagen fibril Collagen fibres
What is scurvy?
Scurvy is a connective tissue defect in the gums due to a lack of strength
What is scurvy caused by?
Lack of vitamin C so is an example of a disease caused by environmental influence.
Vitamin C is a co-factor required for prolyl hydroxylase to function. Therefore, people with vitamin C deficiencies have few prolyl hydroxylases functioning, less proline residues are hydroxylated in the ER during the chemical modification of pro alpha chains, so less hydrogen bonds are present between the alpha chains in the resulting tropocollagen
What does Lysyl oxidase do and which co-factors are required for it to function?
Copper 2+
Vitamin B6
Required to form cross-links between the tropocollagen to form collagen fibrils
What does prolyl hydroxylase do and which co-factors are required for it to function?
Catalyses hydroxylation of proline residues in pro alpha chains
Fe2+ and vitamin C
What would a vitamin B6 deficiency cause?
Less functioning lysyl oxidase as it is a co-factor for this enzyme
Less cross links between tropocollagen to form fibrils
Weaker collagen fibrils
What is the molecular formulation of type II collagen and where is it found?
3 alpha 1 chains
Cartilage, intervertebral discs
What is the main difference between type 1 and type 2 collagen?
Type 2 collagen fibrils do not aggregrate form collagen fibres
Type 1 collagen fibrils aggregate to form collagen fibres
Where is type III collagen found? What is its molecular formular?
Reticular fibres- provide a supporting framework/sponge
Present in the cardiovascular system, around nerve and muscle cells
3 alpha 1 chains
Where is type 4 collagen found? What is its molecular formula?
Basement membrane. 2 alpha 1 and 1 alpha 2 chain
What causes Ehlers-Danlos syndrome and what are the symptoms?
Mutation in collagen type V or Lysyl oxidase deficiency
Stretchy skin
What are the physiological roles of haemoglobin and myoglobin.
Haemoglobin -transporter for oxygen
Myoglobin-store for oxygen
What is the difference between the oxygen saturation curves at different partial pressures of oxygen for myoglobin and haemoglobin? Why?
Myoglobin - hyperbola due to no cooperative binding
Haemoglobin - sigmoid due to cooperative binding
Why does cooperative binding occur in haemoglobin and not myoglobin?
There are four different subunits in haemoglobin as it is made up of 2 alpha chains and 2 beta chains with 4 heme groups, each in one sub-unit. Myoglobin has only one polypeptide with one heme group. Therefore, binding of oxygen to on heme group in haemoglobin causes a conformational change in the protein making the other heme groups more accessible.
How does the binding of oxygen change the conformation of haemoglobin?
Binding of oxygen to a heme group pulls the Fe2+ up into the plane of the protoporphyrin ring pulling the proximal histidine residue that is attached on the other side up. This increases stability of the R state (high affinity state) where there are no hydrogen bonds between hystidine and aspartate residues from the other polypeptides.
What is the R state and T state in haemoglobin?
R state- high affinity, no interactions between histidine and aspartate residues of different polypeptides
T state-low affinity, interactions between histidine and aspartate residues from different polypeptides
Why is cooperative binding important for the physiological role of haemoglobin?
Allows it to be a good transporter of oxygen.
At high PO2 in the lungs, haemoglobin exists in R state and has a high affinity for oxygen so becomes saturated with oxygen.
At low PO2 in tissues, haemoglobin exists in T state and has a low affinity for oxygen so becomes unsaturated with oxygen.
What is BPG and how does it affect haemoglobin
It is an allosteric inhibitor for haemoglobin so shifts the oxygen dissociation curve to the right in order to increase stability of the T state so the haemoglobin has a lower affinity for oxygen.
It is negatively charged so interacts with histidine residues.
What effect do CO2 and H+ have on haemoglobin?
CO2 and H+ decrease pH –> Bohr effect, shifts curve to right
They protonate the negatively charged residues in haemoglobin and promote the CO2 covalently bonds to the N terminals of the polypeptide chains in haemoglobin. This increases the stability of the T state so haemoglobin has a lower affinity for oxygen in respiring tissues.
What effect does CO have on haemoglobin?
Binds more tightly to heme groups than oxygen
When present on some heme groups it increases affinity of haemoglobin for oxygen.
When present on all heme groups, no oxygen can bind.
Shifts curve to left
Which way do allosteric activators/allosteric inhibitors shift the haemoglobin dissociation curve?
Activators-left to increase affinity
Inhibitors-right to decrease affinity
What is the difference between fetal haemoglobin and adult haemoglobin?
Fetal haemoglobin - 2 alpha and 2 gamma chains
Higher affinity for oxygen than adult haemoglobin
Less affected by BPG as there as serine replaces histidine in gamma chains
What is sickle cell anaemia caused by?
Single base substitution, missense mutation
Changes negatively charged glutamate to valine
Hydrophobic residue replaces hydrophilic residue that used to be on the outside of the protein so haemoglobin dimers form as the valines from different molecules interact to block out water
Sickling of red blood cells
How does the sickle cell shape of red blood cells affect their function?
Blocks capillaries, ischaemia, tissue death
Spleen destroys sickle cell red blood cells faster than they can be replaced by bone marrow leading to anaemia
How is sickle cell anemia inherited?
Recessive manner. Shows codominance so heterozygotes have sickle cell trait (some normal and some sickled red blood cells).Sickle cell trait is protective against malaria.
What is thalassemia and how does this differ from haemoglobinopathies?
Haemoglobinopathies-incorrect shape of haemoglobin
Thalassemia-insufficient production of certain globins
What are the different ways in which enzymes can be regulated?
Short-term 1. Substrate and product conc 2. Change in enzyme conformation Allosteric regulation - reversible Chemical modification eg. Phosphorylation -reversible Proteolytic cleavage - irreversible 4. Long-term 1. More enzyme produced/less enzyme produced 2. Enzyme destroyed
Do allosteric enzymes follow Michaelis-Menten kinetics?
No, they have a sigmoidal relationship between substrate concentration and rate of reaction
Allosteric inhibitors/activators increase proportion of which state:
R state or T state
Activators- increase proportion of R state- high affinity so shift curve to left
Inhibitors- increase proportion of T state- low affinity so shift curve to right
Give an example of an allosteric enzyme.
Phosphofructokinase which controls rate of glycolysis
Allosteric inhibitors- ATP, citrate, H+
Allosteric activators- AMP, fructose-2,6-biphosphate
What do protein kinases do?
Transfer the terminal phosphate from ATP to -OH of serine, threonine and tyrosine residues
What do phosphotases do?
Reverse the effect of kinases
Remove phosphoryl groups
Does phosphorylation activate or deactivate enzymes?
Can do either
Why is phosphorylation effective in enzyme regulation? (5)
Links energy status of cell to metabolism. More ATP, more phosphorylation
Allows amplification by cascade reactions
Reversible
A phosphoryl group can make hydrogen bonds, is negative so can change conformation of the enzyme
Rate of phosphorylation can be controlled
Is regulation of proteins by proteolytic cleavage reversible or irreversible?
Irreversible
Give examples of proteins whose action is regulated by protealytic cleavage.
Digestive enzymes Hormones Insulin Collagen Proteins involved in apoptosis
What prevents zymogens from being protealytically cleaved all the time?
Endogenous inhibitors
Give an example of a metabolic pathway in which regulation is controlled mainly by protealytic cleavage.
The clotting cascade
Describe regulation of the clotting cascade.
- Inactive zymogens present in low concentrations.
- Protealytic cleavage of the first zymogen
- Amplification by the cascade effect.
- Clotting factors accumulate at site of damage
- Feedback activation by thrombin ensures continuation of clotting as it acts as an allosteric activator
What is the gla domain present in clotting factors?
Carboxylated glutamate residues that increase the negative charge. This makes them attracted to Ca2+ at the site of damage.
Describe how protealytic cleavage causes the formation of a fibrin clot.
Kringle domains on prothrombin are cleaved.
Thrombin formed at site of damage
Thrombin cleaves polypeptide N terminals in the central globular domain of fibrinogen.
Fibrin monomers can bind to form a clot
Cross links formed between lysine and glutamate residues in different monomers to form hard clot
What shifts the oxygen dissociation curve to the right?
Co2 Acidity BPG Exercise Temp
Why are errors in DNA replication more important than errors in DNA transcription and RNA translation? (3)
The long-term effects of errors in DNA transcription and RNA translation are much less severe because:
- cell makes multiple copies of RNA so unlikely to make same mistake again
- RNA is quickly degraded
- RNA’s are not inherited molecules passed down from generation to generation
What is a mutation?
A heritable alteration in a gene or chromosome but also the process that produces the alteration so are the source of most alleles.
Mutations in which types of cells are not passed on to offspring?
Somatic cells.
Mutations can be dominant or recessive. Describe what these usually result in.
Recessive
Loss of function. Often affect biochemical pathways.
Dominant
Increased function. Often cause structural abnormality.
What are the endogenous sources of mutation? (3)
- DNA replication defects
- transposable elements
- free radicals produced by immune system and mitochondria
What are exogenous sources of mutation? (3)
Ionising radiation (x-rays, uv)
Mutagenic chemical agents/anti-cancer agents
Infection by viruses
Substitution of a single base can be what types of mutations?
Missense - change in amino acid
Nonsense - production of stop codon UAA UGA UAG
Synonymous - codon still codes for same amino acid
What does a substitution in a promoter or enhancer sequence cause?
Amount of gene product altered
Why do mutations occur in germ cells?
There are lots of chances for genetic and chromosomal mutations. Lots of mitosis and meiosis occurs to produce them.
Where can mutations cause greater degradability of mRNA?
Mutation in termination sequence. PolyA polymerase does not recognise this so endonuclease does not cleave the mRNA here and polyA tail is not added
A G>A change in exon 2 of the PAX3 gene that results in a valine 60 to methionine mutation in the gene product.
What molecular technique can detect this?
PCR amplification followed by DNA sequencing
PCR followed by hybridisation with an allele specific probe
Differential gene expression of alternative splicing variants of a given human gene on chromosome 2.
Method(s) of choice for detection. Why?
Microarray
RT-PCR then check size of product by gel electrophoresis
What is southern hybridisation?
- Digest DNA with restriction enzymes.
- Separate by gel electrophoresis
- Hybridise with labelled gene probe
- Detect hybridisation
What is northern hybridisation?
Used to investigate level of gene expression
Same as southern transfer but with mRNA
Which technique can be used to measure the amount of troponin I after a myocardial infarction?
Eliza- antibodies bind to antigen on troponin I. Second antibody linked to specific antibody. Substrate added and converted by enzyme into coloured product.
What is western blotting?
Proteins have been separated by size using protein gel electrophoresis.
Antibodies specific to one protein bind to proteins on blot
Detect binding of this antibody by a second antibody in immunoblot
This indicates whether this protein was expressed in the original sample
What is the difference between western blotting and ELIZA?
Western blotting will allow us to identify the protein
ELIZA will allow us to measure the concentration of the protein
There is a substitution mutation. Which molecular diagnosis method can be used to detect this?
PCR amplification followed by DNA sequencing
PCR amplification followed by hybridisation with an allele specific probe
If the length of a protein has changed due to a mutation, which molecular technique can detect this?
2D-PAGE
Protein gel electrophoresis followed by western blotting
RT-PCR followed by gel electrophoresis.
How can deletion of several contegious genes on one copy of chromosome 17 be detected?
FISH and chromosome painting
Microarray
2D-PAGE (Less proteins present here)
A duplication of one or more exons in the dystrophin gene in a boy with Duchenne Muscular Dystrophy (DMD).
Method(s) of choice for detection. Why?
Southern blotting
Western blotting
2D-page
How can we detect if exons have been deleted?
Southern hybridisation
Amplify with PCR and check size of product by gel electrophoresis
Western blotting
2D-PAGE
How can you detect multiple triplet repeats that occur in Huntington’s using molecular techniques?
Southern hybridisation
What can cause aneuploidy?
Non-disjunction during meiosis or mitosis
Anaphase lag
Why might someone undertake a cytogenetic test?
Diagnostic testing - to account for abnormal phenotype
Prenatal diagnosis - abnormal ultrasound
Predictive testing - prognosis for leukaemia patients
Carrier testing - assess future reproductive risks
What are the different methods of prenatal diagnosis and when are they taken?
Chronic villus sampling, 1st trimester
Amniocentesis, 15 weeks onwards
Why might a mother undertake prenatal diagnosis?
Abnormal first trimester screening
Maternal serum for downsyndrome abnormal
Family history of chromosome abnormality
What is the only monosomy that is viable and why?
Turner syndrome, 45, X
Usually only one X chromosome is ever active in an individual. In a female, the second X chromosome is inactivated. However, there are abnormalities because because there are common genes in the X and Y chromosome where 2 alleles are usually present.
If a mutation causes a change of base from purine to purine, what is this called?
Transition (purine to pyrimadine or vice versa is transversion)
What type of chromosomal mutation is an inversion?
What are the different types?
A segment in a chromosome is rearranged end to end
Pericentric-inversion occurred in a section that encompasses the p and q arm
Paracentric-inversion occurred in a section that encompasses only one of the p or q arm
What is a chromosomal translocation?
A chromosomal segment is moved from one chromosome to another.
What is reciprocal translocation?
Segments from two different chromosomes are exchanged between non-homologous chromosomes. This is a 2 break rearrangement caused by double strand breaks.
Balanced chromosomes-all genetic material present in cell
Unbalanced chromosomes- not all genetic material is present in the cell- some regions of trisomy and monosomy.
What is a Robertsonian translocation?
Two acrocentric chromosomes are fused together (13 14 15 21 22).
Translocations between other chromosomes occur but these gametes do not produce viable offspring.
What can be the different outcomes of meiosis in a person who has a reciprocal translocation?
Alternate = gamete has unbalanced chromosomes with all genes present. Alternate non-homologous centromeres segregate together.
Adjacent 1 = gamete has unbalanced chromosomes with some regions of trisomy and some regions of monosomy. Adjacent non-homologous centromeres segregate together.
Adjacent 2 = gamete has unbalanced chromosomes with lots of regions of trisomy and monopsony. Adjacent homologous centromeres segregate together.
In individual has a homologous 21 Robertsonian translocation. Predict what will be present in the phenotype of all of their offspring?
Downs syndrome
Trisomy 21 is always present
In what circumstances is a quadrivalent formed at meiosis?
Reciprocal translocation
In what circumstances is a trivalent formed at meiosis?
Robertsonian translocation
An abnormal newborn is monosomic for the terminal part of the long arm of chromosome 13, but otherwise has normal chromosomes. The abnormality could be due to…
A) an unbalanced segregation of a balanced Robertsonian 13;13 translocation carried by the mother.
B) an unbalanced segregation of a balanced reciprocal 8;13 translocation carried by the father.
C) loss of a chromosome 13 by anaphase lag in one of the early mitotic divisions in the embryo.
D) chromosome breakage in the oocyte.
D
What are potential treatments for sickle cell disease?
In normal adults, regulatory repressor proteins turn off gamma globin so fetal haemoglobin is not produced. By preventing the production of this regulatory repressor protein, fatal haemoglobin would continue to be produced.
Or
Genetic editing by crispR
What are symptoms of sickle cell disease?
Severe pain
Fever
Anaemia
Associated with sudden death
When is the usual age of onset of Huntington’s disease?
40-50 years.
More CAG repeats associated with earlier onset but large variation so multifactorial-other genes are involved.
As it is passed down generations, earlier onset (anticipation)
Chromosomal mosaics have 2 or more chromosomally different populations of cells (cell lines) in their bodies. Possible causes include…
A) mosaicism in one parent.
B) non-disjunction in the formation of one of the gametes.
C) two sperm fertilising a single egg.
D) non-disjunction in one of the early mitotic divisions of the zygote.
D
A 44 year old man called Jack and his 42 year old wife called Jill are expecting a baby. They have come to hear the results of a routine prenatal screening Jill had some weeks ago. The test results show that the foetus is a female with Down syndrome. There is no family history of genetic disorders in either family. What the most likely genetic cause is of the fetus’s condition and what other possibilities can you think of. Explain in detail.
As there is no family history of genetic disorders trisomy caused by translocations is unlikely. Due to the maternal age the most likely genetic cause is non-disjunction during meiosis I in the mother. In females primary oocytes enter meiosis I before birth, and remain arrested in meiosis I until ovulation. In Jill’s case the primary oocyte was in meiosis I for 42 years, which is a very long time in which environmental agents may have damaged the cell.
Other possibilities are non-disjunction in meiosis II in the mother and of course non-disjunction in meiosis I or II in the father.
A technician from a genetics laboratory shows you a picture of the result of an in situ hybridisation experiment using a fluorescent probe specific for chromosome 13. On the picture you can see interphase nuclei with three fluorescent spots. Give two possible explanations for this.
(1) trisomy 13, i.e. 3 copies of chromosome 13 makes 3 fluorescent spots.
(2) one normal chromosome 13 (one fluorescent spot) and one reciprocal translocation of chromosome 13 with another chromosome (which makes the other two spots, this is because the breakpoint of the translocation on the chromosome 13 in in the middle of the FISH probe that was used).
An individual has a balanced reciprocal translocation. A clinical report is being made to highlight reproductive risk. What type of additional analysis does the scientist need to do before writing a clinical report? Describe some of the possible outcomes of this additional analysis.
Segregation analysis to see what might happen in meiosis. Segregation patterns to consider are:
Alternate segregation that will produce both normal and balance gametes Adjacent 1 segregation that will produce unbalanced gametes Adjacent 2 segregation that will produce unbalanced gametes
What is uniparental disomy? What are its consequences?
Inheriting a pair of homologous chromosomes from one parent. No consequences if the chromosome involved is not imprinted. If imprinted, chromosomes will show differential expression of specific genes depending on the parental origin of the chromosome.
What is the difference between isodisomy and heterodisomy?
Isodisomy=2 identical chromosomes from one parent
Heterodisomy=2 homologous chromosomes from one parent
How do heritable changes in gene expression occur without entailing changes in DNA sequence?
- Differences in the rate of methylation and acetylation of the histones. Methylation-deactivated euchromatin. Acetylation-activated euchromatin
- Non coding RNA involved in de-activating mRNA
- modifications to histones
This is known as epigenetics
Which gene is mutated in many cancers?
P-53 tumour surpressor
What causes emphysema?
Deficiency of alpha 1 antitrypsin caused by smoking
This causes destruction of alveolar walls by elastase
What ensures continuation of blood clotting?
Thrombin activates the intrinsic pathway by positive feedback so the cascade of proteolytic activation of factors continues
What stops blood clot formation?
- localisation of prothrombin-factors are diluted by movement of blood and removal by liver
- proteolytic activation-thrombin binds to thrombomodulin on the endothelial membrane activating protein C to break down factors
- inhibitors-anti-thrombin III enhanced by heparin binding
What causes clot breakdown?
Streptokinase and t-PA stimulates the conversion of plasminogen to plasmin. Plasmin activates the conversion of fibrin clots into fibrin fragments.
In which chromosomes can robertsonian translocations occur?
Acrocentric-13 14 15 21 22
What is turner syndrome?
45, X
Caused by non-disjunction or anaphase lag
Usually one X chromosome is ever active in a human cell as X inactivation occurs in females and males have X and Y. A single X chromosome is a problem because X and Y chromosomes have short regions in common and for normal gene expression 2 alleles of the genes found on these chromosomes are required
What is edwards syndrome?
Trisomy 18
What are the different centromere positions?
Metacentric- in the middle of the chromosome
Submetacentric- between the middle and end
Acrocentric- between submetacentric and telomeres
What is respiratory alkalosis and what causes it?
Cause: hyperventilation
More Co2 expelled than usual. Lower concentration of co2 in alveoli than usual. Steeper conc gradient between capillaries and alveoli. Lowers concentration of co2 in blood. Decreased carbonic acid. Equillibrium shifts to increase carbonic acid conc by decreasing carbonate and hydrogen ion conc. Increases blood pH.
What does carbonic anhydrase do?
Catalyses
H2O + co2 —> h2co3
Which DNA repair mechanism repairs single strand breaks?
Base excision repair
Which DNA repair mechanism repairs bulky adducts and pyrimidine dimers?
Nucleotide excision repair
What is the pH of a lysosome and how is this maintained?
4.5-5. Proton pump
What is the autosomal dominant pattern of inheritance if both parents have the disease?
75%
How is duchenne muscular dystrophy inherited? Is it unifactorial or multifactorial?
x-linked recessive
Unifactorial
Which gene is responsible for Huntington’s disease?
HTT gene
What different phenotypes are present in the ABO blood group system?
Different antigens on red blood cells
What are the different types of enzyme inhibition?
- Reversible - competitive and non-competitive
2. Non-reversible
What is penetrance in autosomal dominant diseases?
The proportion of heterozygotes who express a trait even if mildly
Erythromyosin is an antibiotic that targets 50s ribosomal subunits. What does this mean?
Affects prokaryote ribosomes. Not eukaryotic ribosomes. Does not affect translation in humans but kills bacteria by preventing translation.
What is the UTR in mRNA?
Regions in the mRNA that are not translated. Before AUG start codon and after UAA/UGA/UAG stop codon.
Where is myoglobin found? Why?
Cardiac and skeletal muscle
There is a high demand for oxygen here
What is the difference between alpha thalassemia and beta thalassemia?
Alpha thalassemia-absence or decreased production of alpha globin.
Beta globin molecules form tetramers with a higher affinity for oxygen than haemoglobin.
Beta thalassemia-absence or decreased production of beta globin.
Alpha globin molecules cannot form tetramers
What is protein disulphide isomerise and where is it found?
In the ER. Ensures disulphide bonds have formed in the correct places.
Why is uni-parental disomy rare?
It is the result of 2 abnormal events. Caused by:
- trisomy rescue
1. Non-disjunction in parents meiosis (one extra chromosome)
2. Anaphase lag in foetus (removes extra chromosome) - monosomy rescue
1. Non-disjunction/ aneuploidy in parents meiosis (one less chromosome)
2. Non-disjunction in foetus (adds a chromosome) - gamete complementation
- mitotic error
At what stage in the cell cycle are cells obtained for karyotyping?
Metaphase
What is gene fusion?
Give an example of a disease caused by gene fusion.
Reciprocal translocation occurs between 2 non-homologous chromsomes.
Segment of chromosomes have been moved from one chromosome to another.
The new sequence of DNA has resulted in 2 genes being so close together that the genes fuse to produce a new gene.
Eg. Leukaemia
Between chromosome 9 and 22
Gene fusion creates gene for tyrosine kinase
Gene for tyrosine kinase usually highly regulated but now it is active all the time
Tyrosine kinase is important in growth, differentiation, apoptosis, metabolism
How is the structure of the SRP receptor related to its function?
Composed of 6 proteins and a short piece of rRNA
rRNA -allows it to recognise free ribosomes and the signal sequence while the protein is being translated. The signal sequence is found on the N-terminal so this occurs shortly after translation begins.
The SRP receptor can then direct the protein to the membrane.
Proteins -when an SRP receptor-ribosome complex is formed, it has a complementary shape to the translocon on ER membrane
