Exam questions Flashcards
Why do diseases caused by mutations in the mitochondrial genome vary so much within
families in terms of severity?
-Mitochondria (some wild type, some mutant) that are maternally inherited and the distribution
in an a particular oocyte is variable. Thus within a family this causes variability
-During development the distribution of mitochondria between different tissues will vary and
the proportion of mutant mitochondria will be different in different lineages
-Some tissues with high energy demand that contain higher proportions of mutant mitochondria
are more likely to show pathology than others and this effect will also be variable in family
members.
Identify THREE factors that may prevent an enzyme from achieving its maximum catalytic
capacity (kcat) in vivo, briefly describe how each influences enzyme activity.
Sub-saturating concentration of substrate – failure to maximise concentration of ES complex
Presence of inhibitor (either a simple competitive inhibitor or allosteric effector) –
compromises production of ES complex (inhibits binding of substrate or prevents formation
of active form of enzyme)
Presence of finite concentration of product (a special case of above) – prevents substrate
binding by competitive inhibition, and/or decreases net forward activity by catalysing the
reverse reaction
Sub-optimal concentration of essential coenzyme, such as ATP or NAD (or sub-optimal ratio
of ATP/ADP or NADH/NAD+
) – similar to above (i and iii), this is effectively a sub-satruating
concentration of co-substrate for the reaction and/or sub-optimal concentration of coproduct
of reaction
Covalent modification such as phosphorylation – not all of enzyme in most active form
Incomplete proteolytic processing of an inactive zymogen – prevents enzyme from adopting
its final active conformation
pH – suboptimal pH may influence conformation of enzyme, protonation state of critical
catalytic or reactant-binding residues, or ionisation sate of substrate – decreases effective
concentration of active form of enzyme and/or the precise ionic form of substrate
Describe the essential features of TWO different methods for transforming mammalian cells
– one method should be suitable for transient transformation, and the other should be
suitable for stable transformation
-various transformation methods were introduced in the lectures (CaPO4
precipitation/transfection, electroporation, microinjection, mammalian viral vectors)
-Good examples for transient expression might be CaPO4 precipitation/transfection.
Electroporation (noting these can be used for stable integration if selectable marker is
employed)
-stable transformation might be as above, or involve microinjection or use of an integrating viral vector (e.g. lentiviral vectors were discussed in detail in the lectures)
Briefly describe how the accumulation profile of a protein of interest, using an antibody
directed against the protein.
-expect answer to focus on western blotting, as this method was described in detail in the lectures;
some might also mention in situ methods
-answer should include descriptions of: (1) polyacrylamide gel electrophoresis, (2) electroblotting
and (3) detection using primary and secondary antibodies, with the secondary antibody linked to an
enzyme such as alakaline phosphatase
What is DNA methylation?
Cytosine methylated in DNA
Methylation may be symmetric or asymmetric
Symmetric DNA generally constitutively methylated by methylases, DNMT1 in animals or
MET1 in plants
Nucleosome histones modified in their tail regions, affecting DNA compaction and thus
transcription.
What are histone tail modifications?
Modification may be methylation, acetylation, phosphorylation, ubiquitylation
Typical histone ‘marks’ are H3K27Me (transcription repressive), and H3K4Ac
(transcription encouraged).
Histone modification effected by specific enzymes, e.g.
Histone methyl transferase.
What is RNA directed gene silencing?
Transposons and other repeated sequences silenced by RdDM (RNA directed DNA
methylation)
Involves processing of ‘target’ transcript by Dicer and Argonaut proteins
Cleaved transcript + Argonaut protein binds to gene and encourages methylation.
Evolved originally as viral defence.
What are the ANT-C (Antennapedia Complex) and the BX-C (Bithorax Complex)?
Clusters/complexes of Hox genes. Hox genes tell cells where they are along head to tal axix. Expect
them to say they are in Drosophila. (Technically only in Drosophila melanogaster and some close
relatives, bceuase in other flies and other animals, the genes are arranged differently, but that is
getting a bit too technical)
Draw a diagram showing how the signal recognition particle (SRP) assists in the translocation
of proteins from the cytosol to the endoplasmic reticulum?
o SRP Binds to a signal sequence at the N terminus of a protein emerging from the ribosome
and stops translation.
o Transfers the stalled ribosome to a ribsome receptor on the rough endoplasmic reticulum
(ER) by binding to an adjacent SRP receptor.
o Hydrolysis of GTP releases the SRP from the ribosome allowing protein synthesis to resume
with concurrent transport of the protein into the ER via a peptide translocation complex in
the membrane.
o Overall energetics: Free ribosome + nGTP → Bound ribosome + nGDP
What is heritability? How can heritability be estimated? What does a heritability of
zero mean?
-The proportion of the phenotypic variance that can be explained by additive genetic
effects
-Regressing offspring trait values against parental trait values. The heritability is the slope
of the fitted regression line
-There is no additive genetic variance for the trait in the environment tested.
Why does the genetic map distance between two loci correlate poorly with their physical
map distance?
- The genetic map distance is calculated from the recombination frequency between the two
loci, physical distance: number of base pairs between the two loci. - recombination rate varies along the chromosomes: recombination hot spot and cold spots,
low recombination frequency at centromere. - Double ( or any even number of) cross overs between the two loci are not detected in the
progeny, hence further apart the genes are the more recombination frequency
underestimates the physical map distance.
Name the fibres providing tensile strength in the extracellular space of animals and plants.
List TWO major differences between them.
Animals: collagen, plants: cellulose
Differences:
1, collagen fibril is a protein polymer (triple helix collagen protein subunit)
cellulose is a polysaccharide ( D-glucose monomers)
2, collagen protein helixes are synthesized in endomembrane system (ER- Golgi-secretary vesicle)
and secreted into extracellular space.
cellulose is synthesized at plasma membrane by cellulose synthase (rosette) complex
3, Cellulose forms crystalline microfibrils
Collagen does not form crystalline structure.
Outline THREE characteristics typical of eukaryotic centromeric DNA
- Much of it repetitive in sequence
- Transposon-rich (heterochromatic; particularly rich in retrotransposons)
• Gene-poor (low density of active genes – also a characteristic of
heterochromatin)
