General

Question 1

Phospholamban
- gene and protein
- function

Answer 1

• encoded by PLN gene
• NB in lusitropic effect in heart
• substrate of cAMP-dependent protein kinase (PKA)
• unphosphorylated state inhibits SERCA2, phosphorylation by PKA disinhibits SERCA - allows faster Ca uptake into SER

Question 1

What are scaffold proteins

Answer 1

Proteins NB in signalling pathways in cells - binds to members of signalling pathways and tethers them to complexes

Question 2

Function of caveolin

Answer 2

Caveolins are scaffolding proteins/integral membrane proteins NB in caveolae membranes and function of receptor-independent endocytosis
- NB in compartmentalising and concentrating signalling molecules

Question 3

Role of ALPK3

Answer 3

• codes for alpha protein kinase 3
• believed to act as transcriptional regulator through activation of cardiac transcription factors

Question 4

Role of junctophilin 2

Answer 4

-encoded by JPH2
- part of junctional complexes NB in mediating cross talk between plasma membrane and ER/SER ion channels

Question 5

What is nonsense-mediated decay
- which proteins involved

Answer 5

NMD is a translation-coupled mechanism that eliminates mRNAs containing premature termination codons (PTCs)
- UPF proteins (UPF1, 2 and 3)
- exonucleases

Question 6

What is cosegregation analysis

Answer 6

Technique to establish inheritance pattern of specific variant within a family - constructs pedigree and establishes whether variant co-occurs with phenotype

Question 7

Mendel’s 3 laws

Answer 7

1- Law of segregation: each individual has 2 alleles, one from each parent, that segregates during gamete formation
2 - Law of Independent Assortment: genes from different chromosomes assort independently during gamete formation
3- Law of Dominance: in heterozygous individuals, 1 allele (dominant) will determine phenotype and recessive allele has no noticeable effect on phenotype

Question 8

Definition of linkage analysis

Answer 8

Statistical method of mapping genes for heritable traits to their chromosomal locations
- genome-wide markers (microsatellites or SNPs) tested in pedigrees segregating a trait
- using statistical method (of linkage analysis) combines data to determine chromosomal regions likely to harbour genes for trait
- Parametric linkage analysis used for Mendelian traits (vs model-free LA for complex traits)
- LOD and recombination fractions used to test gene locations

Question 9

LOD score in linkage analysis

Answer 9

Logarithm of the odds score
- evaluates if 2 loci (location of genetic markers) are genetically linked, ie inherited more often than expected by chance

Question 10

Examples of chromatin annotations

Answer 10

• Histone modifications (H3K4me3 (associated with active promoters), H3K27ac (associated with active enhancers), and H3K9me3 (associated with heterochromatin)
• DNA methylation (at CpG dinucleotides - gene silencing)
• Open chromatin (eg ATAC-seq - identifies regions of open chromatin - assoc with active gene expression)
• Promotor and enhancer regions
• Transcription binding sites
• Topologically associating domains (TAD)

Question 11

Initiative (projects) for chromatin mapping

Answer 11

• ENCODE (encyclopedia of DNA elements)
• Roadmap Epigenomics
• BLUEPRINT project

Question 12

What are costameres
- 2 complexes it consists of

Answer 12

Structural-functional component that connects sarcomere to sarcolemma
- DGC (dystrophin-glycoprotein complex)
- Integrin-vinculin-talin complex

Question 13

What are the protein structures in the DGC (dystrophin-glycoprotein complex)

Answer 13

• Dystroglycans
• Sarcoglycans
• Dystrophin
• Sarcospan
• Syntrophin

Question 14

Function of costamere

Answer 14

• Keeps sarcolemma in line with sarcomere during contraction
• lateral (perpendicular) transmission of force generated by sarcomere to sarcolemma and ECM

Question 15

What is dysferlin

Answer 15

-Dystrophin associated fer-like protein
- DYSF gene
- sarcolemma repair and calcium signalling
- dysferlinopathies, muscular dystrophies (incl limb-girdle MD)

Question 16

Gelsolin

Answer 16

• Severs F-actin
• Caps barbed ends of actin filaments preventing monomer exchange
• Promotes nucleation step of actin polymerization

Question 17

What are nuclear localisation signals (NLS) and how do they work

Answer 17

• Short sequences of amino acids or motifs on a protein that serves as targeting signal for transportation of protein into nucleus
• Importin-α receptor recognises the sequence and binds protein, then couples onto Importin-β and facilitates transport into nucleus

Question 18

2 primary types of nuclear localisation signals (NLS)

Answer 18

• classical NLS (cNLS): single, continuous stretch of amino acids, typically lysine rich (PKKKRKV) - K=lysine
• bipartite NLS (bNLS): 2 clusters of amino acid sequences separated by short spacer region

Question 19

What are filamin proteins
- which NB in heart/muscles

Answer 19

Filamin are class of proteins that holds 2 actin filaments at large angles
- Filamin C NB in cardiac and skeletal muscles, contribute to Z-disc proteins

Question 20

What is vinculin

Answer 20

-membrane cytoskeletal protein in focal plaques involved in the linkage of integrin adhesion molecules to actin cytoskeleton
-anchors F-actin to membrane
- binds alternatively to a-actinin or talin

Question 21

What are focal adhesions (FA)

Answer 21

• Large macromolecular assemblies through which mechanical force is transmitted between the ECM and interacting cell.
• Subcellular that mediates signalling events from outside (in response to ECM adhesions/substrate)

Question 22

What are chimeric proteins

Answer 22

• AKA fusion proteins or hybrid proteins, created by combining genetic sequences of 2 or more different genes or proteins
• Can be natural (eg from chromosomal translocations) or engineered

Question 23

Examples of engineered chimeric proteins

Answer 23

• Reporter proteins (reporter gene or fluorescent protein): allows tracking of expression and localisation
• Therapeutic proteins: eg antibody-drug conjugates (ADC) in cancer
• Enzyme fusions: biocatalysis - multiple enzyme reactions in a single protein
• Antibody engineering: combining parts of Ab from different species

Question 24

2 types of actin

Answer 24

1. G-actin: globular actin, monomeric form of actin in cytoplasm that can polymerise to form F-actin. Involved in cellular processes and has binding sites for ATP 2. F-actin: filamentous actin. Formed from G-actin polymerisation into linear, helical form. Dynamic structure, constantly undergoing assembly/disassembly in response to cellular signals and needs

Question 25

Some functions of F-actin

Answer 25

- Cell shape and motility - Muscle contraction - Cell adhesion (focal adhesions) - Endocytosis and exocytosis - Intracellular transport - Cell divisions - Neuronal function (maintains dendritic spine structure, NB for neuronal plasticity)

Question 26

How does the yeast two-hybrid system work (Y2H)

Answer 26

- Process of studying protein interaction where a transcription factor is split into 2 parts: DNA binding domain (DBD) and Activation domain (AD) - 2 proteins of interest are fused onto these 2 domains respectively - If the protein interacts with each other, the reconstituted transcription factor becomes active and initiates transcription of a reporter gene in the yeast host - Results in phenotypic change: growth of yeast cell

Question 27

Mechanisms of off-target edits in CRISPR

Answer 27

- sgRNA - recognises a "seed sequence" of 5 bps proximal to PAM that determines specificity (ie longer seed + PAM would've decreased chances of off-target binding) - non-specific PAM binding: recognises NRG (where R is G or A) - Cas9 protein delivery: better with direct delivery as it degrades after cleaving DNA. Plasmid delivery increases off-target edits

Question 28

3 ways in which first strand cDNA synthesis can be primed

Answer 28

using: - Random primers - oligo(dT) - gene-specific primers (GSPs)

Question 29

What is the 3' bias in cDNA synthesis

Answer 29

When using oligo(dT), 3' bias can occur due to reverse transcriptase (RT) efficiency or RNA fragmentation. oligo(dT) = short sequences of deoxythymidine. Binds to poly-A tails of RNA (i.e. the 3' end), ie "primes" cDNA synthesis from the 3' end. The RT process may not always reach the 5' end of RNA, leading to biased representation of cDNA library (ie RT efficiency). RNA fragmentation (due to sample processing or unintentional degradation) will also cause bias (ie RT will work from the 3' end until the fragment site)

Question 30

What are the factors about amino acid variations increase the likelihood of their deleteriousness?

Answer 30

- variations at conserved sites more likely to cause functional changes - variations affecting: -- active sites -- interaction sites -- solubility -- protein stability

Question 31

What are PolyPhen and SIFT scores

Answer 31

In-silico predictors of SNPs/amino acid changes on protein function. - PolyPhen = Polymorphism phenotyping (amino acids) - SIFT = Sort Intolerant From Tolerant (SNPs)

Question 32

What is nebulin

Answer 32

- actin binding protein localised to thin filament of sarcomeres - coded by gene NEB - large protein 600-900kDa, binds 200 actin monomers, "ruler" for thin filament - causes some nemaline myopathies

Question 33

In Dark et al (LV protocol), what did each of these markers indicate: - TNNT2 - MYL2 - HAND1 - NR2F2 - HOXB2

Answer 33

- TNNT2 - troponin T = pan-cardiac marker - MYL2 - myosin light chain 2 = pan-ventricular marker - HAND1 - heart and neural crest derivatives expressed 1 = LV marker - NR2F2 - atrial marker - HOXB2 - atrial marker downstream of RA signalling

Question 34

What are the factors mechanistic factors affecting sarcomere power in heart?

Answer 34

- beat rate - sarcomere length (length-dependent activation) - intracellular Ca flux - phosphorylation of titin - Troponin I - MYBPC3 - Myosin regulatory chain