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YEAR 2 - Genetics > Semester 1 > Flashcards

Semester 1 Flashcards

1
Q

Will the transposon DNA sequence be conserved, mutated to scape silencing or degenerate in the human genome over many generations?

A

They will degenerate

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2
Q

What characteristic distinguishes an indel from an insertion or deletion SV?

A

The length, indels are < or equal to 50bp

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3
Q

If 40% of variation in blood pressure is explained by genetic factors how much is explained by environmental factors?

A

60%

G + E = 100
E= 100 - G
100 - 40 = 60

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4
Q

Can monozygotic twins have different sexes

A

NO

Impossible. Sex is determined by X and Y chromosomes.
MZ twins have identical DNA and therefore inherit the same pair of sex chromosomes

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5
Q

A substitution that causes a change in the amino acid sequence is termed…

A

Non- synonymous
Missense
Nonsense

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6
Q

Non coding SNPs have phenotypic effects because they…

A

Overlap non coding genes
Change the structure and state of chromatin
Overlap transcriptional regulatory regions
Overlap post transcriptional regulatory regions

(The ENCODE project identified several classes of biochemical function of non coding DNA. Some of them overlap SNPs)

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7
Q

What do you call a trait controlled by a single gene?

A

Monogenic or Mendelian

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8
Q

What is an allele?

A

A different version of a gene or variant

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9
Q

What is an SNP?

A

A single nucleotide substitution

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10
Q

What is the maximum dosage of the risk allele in a bi allelic SNP?

A

2

The risk allele is the allele that causes disease or increases the height

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11
Q

How many SNPs have been identified in the human genome?

A

> 100 million

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12
Q

What is the minor allele frequency of a SNP?

A

The frequency of the least common allele

Rarest allele in the population

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13
Q

What element covers the greatest part of the human genome?

A

Repetitive and transposable elements

50% of our genome is junk DNA!
Many of the transposons are not working anymore, mutation in enzyme, enzyme loses function and transposon becomes silent.
Silencing RNA can silence them

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14
Q

Select all the structural variants

(Where a long length of DNA has been changed - KEY)

A

Deletion and Inversion

Deletion Means the chromosome has changed as you have less of a sequence now
Inversion means you change the whole structure of large part of chromosome.

Indels are also the same but are not considered a structural variant as are shorter in length - only 50bp.

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15
Q

What is a variable number tandem repeat?

A

A variable end to end duplications of a sequence motif

Variable number because the number of motifs will vary
Tandem repeat, repeat that is in tandem because it’s facing eachother

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16
Q

A polygenic trait is a phenotype controlled by…

A

Multiple variants (loci)

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17
Q

A complex trait is controlled by…

A

Multiple variants and the environment

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18
Q

What is heritability?

A

The proportion of disease risk explained by genetic factors

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19
Q

In Eukaryotes what proteins control gene expression?

A

RNA pol 2 - generates mRNA always

General transcription factors

Transcription factors - increase transcription (co activators)

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20
Q

What do repressors do?

A

They act as inhibitory factors to transcription

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21
Q

What affects the binding of proteins to DNA?

A

If there’s any co factors - having a + charge is beneficial, helps with interaction with - charged DNA backbone

Shape of the DNA / protein

DNA recognition sequence

Mediator contents

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22
Q

What is NOT part of Sanger Sequencing?

A

Multiple distinct DNA fragments

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23
Q

What can give different results according to sampling tissue?

A

RNA sequencing - it is tissue specific!

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24
Q

What is linkage disequilibrium?

A

It’s the correlation between the genetic variants - how likely they are to inherit it together

It is population specific

It diminishes with physical distance - snips that are closer together in the genome are usually more closely linked, so likely to be inherited together

It can be used to select snips for genotyping a race

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25
Q

What is false about polygenic risk scores?

A

They can be used for diagnostic purposes

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26
Q

What would be the most appropriate genetic testing method to confirm the diagnosis of Cystic Fibrosis?

Essay based question!

A

Sanger sequencing

Because it is a single gene, would NOT use next generation sequencing as that is done for whole genome!

Justify and say why not

Introduction about the disease

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27
Q

What is a mediator complex?

A

A multi subunit assembly that appears to be required for regulating expression of most RNA pol 2 transcripts

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28
Q

What is a co activator?

A

A protein or protein complex that activates genetic transcription usually by binding to a transcription factor

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29
Q

What is a co repressor?

A

A molecule that is capable of combining with specific repressor molecule and activating it - thereby blocking gene transcription

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30
Q

What is a dimer?

A

A chemical compound composed of 2 similar subunits or monomers

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31
Q

What is Hetero dimerisation?

A

A complex composed of NON identical components

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32
Q

What is homo dimerisation

A

A complex composed of identical components

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33
Q

What are transcription factors?

A

Proteins that move into nucleus to bind to specific sequences (response elements) and recruit additional proteins to stimulate transcription

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34
Q

What is a consensus sequence?

A

Sequence that comprises the most commonly encountered nucleotides found at specific locations in the DNA

2 main ones are pribnow box (10bp upstream of the TSS) and -35 sequence (35bp upstream of the TSS)

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35
Q

What does non coding mean?

A

DNA or RNA that is NOT translated into protein

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36
Q

What does polycistronic mean?

A

a single mRNA that generates multiple proteins

mRNA is generated from multiple genes

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37
Q

What is a promoter?

A

DNA sequence where transcription apparatus binds to initiate transcription

It indicates direction and which strand of DNA elongation occurs on, also contains TSS

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38
Q

What is the transcription start site TSS?

A

The first DNA nucleotide that is transcribed into RNA

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39
Q

What are transcription factors?

A

Proteins that move into nucleus to bind to specific sequences (response elements) and recruit additional proteins to stimulate transcription

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40
Q

What is false about GWAS?

A

They have discovered ALL of the estimated heritability for most traits

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41
Q

What are examples of precision medicine?

A

Diagnostic tests based on genetics

Gene therapy

Providing the correct dose of a drug based on a persons genetics

Stratifying patients into more similar groups for more targeted treatment and follow up

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42
Q

What is NOT true about Sanger sequencing?

A

It is used for sequencing multiple genes

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43
Q

The plot to describe the results from a GWAS is called…

A

Manhattan plot

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44
Q

Third generation sequencing uses…

A

Unamplified DNA

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45
Q

Linkage disequilibrium…

A

Is the correlation between genetic variants

Is population specific

Usually diminishes with physical distance

Is used to select SNPs for genotyping arrays

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46
Q

What is the length of the human genome?

A

3 billion - 3 000 000 000

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47
Q

How many chromosomes composes the human kariotype

A

46

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48
Q

What is the total physical length of human DNA

A

2m

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49
Q

What part of the gene codes for protein?

A

Exon

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50
Q

How many protein coding genes have been identified in the human genome?

A

20 000

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51
Q

What element covers the greatest part of the human genome?

A

Repetitive and transposable elements

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52
Q

How many SNPs have been identified in the human genome?

A

100 000 000 - 100 million

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53
Q

How many SNPs are different between 2 unrelated individuals?

A

4 000 000

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54
Q

What is an SNP with an impossible number of alleles?

A

Penta allelic

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55
Q

What is an indel?

A

An insertion or deletion with less than 50bp

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56
Q

What is NOT a long structural variant?

A

Indel

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57
Q

What are the least common structural variant m?

A

Translocations

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58
Q

What type of VNTR is most frequent in the coding region of genes?

A

Tri nucleotides

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59
Q

What elements are not part of the 3D organisation of the chromosomes?

A

Nuclear lamina

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60
Q

What DNA facilitates storing, reading and copying biological information?

A

Complementarity

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61
Q

How many transcripts have been identified in the RNA of all cell types investigated so far?

A

200 000

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62
Q

What is the most prevalent family of genes lost in humans when compared with chimpanzee?

A

Olfactory receptors

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63
Q

What gene function is not enriched for ultra conserved elements?

A

Signal transduction

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64
Q

How is gene expression associated with copy number?

A

Directly proportional

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65
Q

What type of gene is most enriched for copy number variants?

A

Non coding genes

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66
Q

What is a detectable output of transcription?

What methods could you use to measure the outputs?

A

Look at DNA binding proteins
Look at RNA

Use PCR to detect the RNA

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67
Q

How do we know if we have the right products for PCR?
PCR may lead to several bands on your gel of different sizes etc

A

How do we know what size we’re looking for?
You’ve got to use your primer sequences!!
Do a BLAST search and it searches for where those primers bind. There will be one at the beginning and one at the end - so find the distance between the two primers!
Need to know how big the product is you’re looking for as BLAST will come up with more than one product

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68
Q

Why may you have a high band on PCR?

A

Sequences that are in the primers (if they’re inside the exons) will also exist in your genomic DNA.
When you harvest cells, you are extracting RNA but some DNA may get through! May end up with some genomic copy which will be much larger because it has all the introns inside it as well as the exons

Could try changing primers to overcome this

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69
Q

What methods could you use to test the theory that p53 increases transcription from the PUMA promoter?
P53 = beta sheet containing transcription factors

Could you tell how much more transcription is happening?

A

Have 2 plates of cells: some with endogenous p53 and some where you’ve added more p53.
Compare and see when you do PCR where the specific primers for PUMA mRNA and see whether there is a more intense band where you’ve added p53 vs the non added one!

NO - looking it on a gel is qualitative, to establish that you would need to use quantitative PCR

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70
Q

How is quantitative PCR beneficial over normal PCR / reverse transcriptase PCR?

A

It monitors fluorescence

Quantitative tells you how much brighter it might be as detector tells you how bright it is in comparison to others

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71
Q

Why would it be a good idea to shut off translation in a situation with low amino acids?

A

When levels are low of certain amino acids you might end up getting use of the wrong amino acid = wrong code for protein. So want to avoid making those proteins that are missing the correct amino acids!
Don’t want to waste energy when we don’t have all the right resources to make the proteins.

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72
Q

Why would it be a good idea to switch off translation when cells are stressed (eg when infected with a virus)?

A

All viruses that infect cells may come in with their own RNA or DNA.
They are totally dependant on the translation machinery of the cell that they’re infecting
So it’s a good line of defence from the infected cell, stops virus infection! If it knows it’s infected, it can shut off protein synthesis = stops production of viral proteins

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73
Q

What could be the consequences of translating a protein with a PTC (premature termination codon)?

A

Protein will be non- functional! Can’t do job properly
Waste of energy

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74
Q

What situation in the cell would we want to turn on GCN4?

A

When amino acids are low in the cell! So need to synthesise more

eIF2 alpha is activated when amino acids are low, it is phosphorylated = a reduction in the amount of available ternary complex for translation initiation

GCN4 helps synthesise amino acids to help get out of this cell stress situation!

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75
Q

What is part of a eukaryotic gene?

A

Exons
Introns
Promoter
Enhancer
5’ and 3’ untranslated region

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76
Q

What is correct about transcription?

A

Transcription is regulated by regions of DNA far away (100s of KB away from transcriptional start site)

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77
Q

What are the molecular mechanisms for mRNA localisation?

A

Random diffusion and trapping and generalised degradation in combination with local protection by trapping - eg Drosophilia mRNAs enriched in pole plasma and posterior pole of the embryo

Directed transport on cytoskeleton - eg neuronal RNPs along microtubuli and ASH1 mRNA along actin filaments to bud tip in yeast

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78
Q

What is required for ASH1 mRNA transport to the bud tip of dividing yeast cells?

A

She2, She3 and Myo4 (Myosin) proteins are required for ASH1 mRNA transport along acting ‘cables’

Khd1p is a translational repressor

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79
Q

Additional proteins are associated with the core (She2-She3-Myo4) RNP complex for …

A

RNA transport

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80
Q

RNA cargo is released via phosphorylation of a translational repressor (Khd1p) at the bud tip.
How does this happen?

A

Finally, the transport machinery is released and enables local translation of ASH1 at the bud tip.

ASH1 protein in daughter cells prevents mating type switching

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81
Q

Khd1 protein co localises with a subset of the bud localised …

A

mRNAs

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82
Q

Xist is a large (17kb) cis acting regulatory IncRNA.
What is X inactivation specific transcript?

A

XIST associates with the X chromosome that it was expressed from (cis regulation)

Initiates Histone modifications (methylation, deacetylation) which results in heterochromatin formation

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83
Q

XIST gene.
What diseases is the unbalanced X expression associated with?

A

Unbalanced X expression involved in diseases associated with infertility and mental retardation

Eg Turner, Rett, Kliefelter’s Syndrome

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84
Q

What are the recurring concepts in post transcriptional gene regulation?

And how is specificity given?

A

Cis acting elements in mRNAs

Trans acting factors

Specificity given through: RBP / complexes that bind to sequence / structural elements in the RNA

ncRNAs in association with RBPs anneal with sequences in the RNA target

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85
Q

What are the cis acting elements in the RNA?

A

Polyadenylation signals in pre mRNAs

Splice sites in pre mRNAs

Regulatory elements in mRNAs influencing translation / localisation

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86
Q

Trans acting factors are specific RNA binding proteins that often act as a scaffold for assembly of larger ribonucleoprotein complexes.

Examples of these?

A

She2 protein (RBPs) as an adaptor for localisation of mRNA to the bud tip in yeast

Iron response protein (IRP) binding to iron response element (IRE) in 5 UTR of ferritin mRNAs

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87
Q

What are examples of the trans acting factors: Ribonucleoprotein complexes?

A

Cleavage and polyadenylation (CPSF, CStF, CF and PAP)

Spliceosome (U1, U2, U4, U5, U6 snRNPs)

Exosome (RNA degradation in nucleus and cytoplasm)

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88
Q

What are examples of target mRNA site selection by base pairing with non coding RNAs?

A

Small nuclear RNAs (snRNAs) base pair with sequences in the pre mRNAs for splicing

miRNAs partially hybridise with sequences in 3 UTR of mRNA target to repress gene expression

Codon anticodon (tRNA) to read the genetic code

Other examples: siRNAs, snoRNAs, IncRNAs

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89
Q

Long non coding RNAs (IncRNAs) are defined as?

A

Being at least 200nts long

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90
Q

Exons are generally -10 times shorter and more uniform than introns.
What is the size distribution of exons?

A

Average length of human exons is 150 bases

On average 10 exons / gene

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91
Q

What is the size distribution of introns?

A

Average length of human introns 1,5 kB

Largest intron is 1,1 Mb (intron 5 in KCNIP4)

Since introns can be very long, additional strategies are required to improve splice site selection!

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92
Q

What is the average length of human exons?

A

150 nts

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93
Q

Different modes for alternative splicing have been observed in:

A

Exon skipping

Intron inclusion

Alternative splice site selection

Alternative transcriptional start site
selection

Mutually exclusive exons

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94
Q

What does cleavage and polyadenylation at the 3’ end of eukaryotic pre mRNAs require?

A

CPSF - cleavage and polyadenylation specificity factor
CstF - cleavage stimulatory factor
Two cleavage factors (CFI, CFII)
Poly(A) polymerase (PAP)

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95
Q

Which of the following factors is NOT involved in the 3 end formation of eukaryotic pre mRNAS?

A

DICER

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96
Q

What is wrong regarding miRNA and siRNA?

A

Both repress translation by imperfect hybridisation with mRNA targets

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97
Q

What is true about both miRNA and siRNA?

A

Both are 20 to 22 nucleotides long RNA molecules

Both anneal with complementary sequences preferentially located at 3 UTR of mRNAs (in complexes with several RBPs)

siRNA may have originated as a defense mechanism

miRNA and siRNA are NOT present in eukaryotes

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98
Q

Small interfering RNAs (siRNAs) cleave mRNAs upon … hybridisation

A

Perfect

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99
Q

How do you break up the cells and separate the RNA from proteins and lipids and other contents of the cells we are not interested in?

(Practical)

A

Resuspend the cells in Lysis Buffer and vortex for 10s to mix thoroughly

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100
Q

Can you amplify genomic DNA using RT PCR (reverse transcriptase pcr) technique?

A

False

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101
Q

To extract RNA, we use a silica membrane. Why?

(Practical)

A

RNA binds to the membrane allowing all the impurities to be washed away and removed

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102
Q

What is the correct use of pipettes?

A
  1. Press until STOP1 to expel air from outside the container
  2. Go into container at 90 degrees and slowly release plunger to draw liquid in
  3. Move the loaded pipette to the desired well and release the plunger to STOP2
  4. Remove the tip from the well and only then release the plunger to resting position
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103
Q

To draw up liquid with a pipette, the correct way is…

A

You hold the pipette perpendicular to the reservoir and touch the liquid as little as you can to avoid carryover, while confirming visually that it is loading correctly

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104
Q

At the beginning of the practical, we need to separate differentiated from undifferentiated MEL cells from culture solution. What is the correct procedure to do this?

A

Put the labelled tubes in the centrifuge, so we can pellet cells at the bottom of the tube and then remove the media

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105
Q

RT PCR stands for…

A

Reverse transcriptase PCR

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106
Q

What do we call a sample in which a known template (RNA or DNA) is added to check if reaction works properly?

A

Positive control

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107
Q

During PCR, what is the stage where we apply high temperatures to separate DNA strands?

A

Denaturation

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108
Q

Our PCR amplicons are designed to cross introns. This means that PCR products are generated from RT PCR will be larger or smaller than the ones from genomic PCR?

A

Smaller

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109
Q

How many master mixes do you need to prepare for genomic PCR?

(Practical)

A

Two

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110
Q

What is true about tRNAs?

A

Deliver amino acids to the ribosome

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111
Q

Which factor binds to the 3’ end of eukaryotic cellular mRNAs?

A

PABP - poly A binding protein

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112
Q

Which protein escorts the tRNA initiator to the 40S subunits?

A

elF2

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113
Q

Which protein unwinds RNA structure to allow 43S scanning?

A

eIF4A - protein with helicase activity that physically unwinds RNA and uses ATP to do it
Can’t do it without 4G and 4E

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114
Q

How can stress impact ternary complex availability? What are the steps?
What are the two outcomes?

A

Stress (amino acid insufficiency, unfolded proteins, viral infections)

eIF2a kinase activation

eIF2a ph - reduced eIF2B available

Reduced eIF2 GTP tRNA (ternary complex) availability

Two outcomes: Global translation downregulation and Selective translation up regulation

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115
Q

What does PERK defect?

A

Unfolded proteins in the ER

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116
Q

What does PKR detect?

A

Viral infections

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117
Q

What does HRI detect?

A

Low heme

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118
Q

What does GCN2 detect?

A

Low amino acids

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119
Q

What does PKR detect?

A

Double stranded RNA - dsRNA

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120
Q

What does PERK detect?

A

ER stress

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121
Q

Why is global translation down regulation helpful?

A

Don’t waste resources

Redirect energy to stress recovery

Don’t make faulty proteins when AAs are scarce

Stop viral protein production

Allow proper protein folding

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122
Q

Why is selective translation up regulation helpful?

A

Produce products to recover from stress eg GCN4 , ATF4

Turn translation back ON (GADD34)

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123
Q

GCN4 is a transcription factor activating more than 50 genes involved in amino acid synthesis.
Is it produced under normal conditions?

A

NO

Ternary complex is abundant under normal conditions

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124
Q

What happens under amino acid stress conditions?

A

uORF translation impaired by eIF2a P and GCN4 translation is promoted

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125
Q

Difference between ternary complex in normal vs amino acid stress conditions?

A

Normal - ternary complex abundant

Stress - ternary complex limiting, only downstream AUG recognised

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126
Q

RNA structure impairs cap binding and scanning. Translation on these structured mRNAs is highly eIF4E dependant.
Unstructured 5 UTR =

Structured 5 UTR =

A

Easy scanning

Tough scanning

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127
Q

eIF4E over expression increases efficiency of…

A

Cap binding

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128
Q

mRNAs with structured UTR include…

A

c myc, VEGF, cyclin D1, FGF

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129
Q

Therapeutic suppression of translation initiation factor eIF4E expression reduces tumour growth without …

A

Toxicity

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130
Q

ASO antisense oligonucleotide can reduce eIF4E expression in … cell lines

A

Human tumour

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131
Q

Which of the following statements about eIF4E is TRUE?

A

The mTOR signalling pathway can regulate eIF4E activity - doing that through the phosphorylation of 4E BP

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132
Q

What is part of a eukaryotic gene?

A

Exons

Introns

Promoter

Enhancer

5’ and 3’ untranslated region

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133
Q

What is correct about Transcription?

A

Transcription is regulated by regions of DNA far from (100s KB away from the transcriptional start site)

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134
Q

What controls transcription in prokaryotes?

A

Operator regions - can recruit transcriptional repressors to promoter

Cis regulatory elements - can recruit transcriptional activators to the promoter

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135
Q

Transcriptional activators ONLY bind to cis elements
True or false?

A

True - in prokaryotes they can only activate transcription

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136
Q

Ligand dependant binding changes the conformation of a protein so that it can bind to DNA.
Are these types of proteins always acting as activators of transcription?

A

NO

Eg Tryptophan was required for binding to the operon
Eg cAMP was required for activator binding to the cis regulatory elements to initiate transcription

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137
Q

What are transcriptional activators?

A

Promotes regulator binding

Recruit RNA polymerase 2

Releases RNA polymerase 2 either to begin transcription OR from a paused state

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138
Q

What are broad promoters?

A

Require assembly of multiple independent protein complexes to form across Kbp of DNA

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139
Q

What are sharp promoters?

A

Controlled by the binding of fewer protein complexes located over a shorter span or non coding DNA

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140
Q

What can affect the strength of a promoter?

A

There can be more than one TSS

There does NOT have to be a TATA box

Chromatin structure can override all of this

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141
Q

What does the term Epigenetics mean?

A

Modification / changes of gene expression that aren’t caused by any changes in the base pair sequence within the genetic code

Includes: condensation and relaxation of chromatin / activators and repressors / chromatin modifiers

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142
Q

What are the components of chromatin?

A

Two molecules EACH of H2A, H2B, H3 and H4 histones. Makes an octamer

146nt of DNA winds 1.65 times around histone core to = nucleosome

Hydrogen bonds form between DNA and histone octamer

Each nucleosome separated from next by 80nt of linker DNA

Histone H1 works as a clamp!

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143
Q

How does chromatin control gene expression?

A

Heterochromatin = short and tightly bound
Euchromatin = long and loose

More tightly wound structure = less access to bases and transcription factors
Folding protects DNA from being exposed for usage

Unfolding will expose DNA to transcription factors = allows promoters to be bound by GTFs and other TF to allow genes to be expressed

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144
Q

Nucleosomes prevent … access by general transcription factors and RNA pol …

A

Promoter
2

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145
Q

Transcriptional activators recruit Coacivators including:

A

Histone modification enzymes

ATP dependant chromatin remodelling complexes

Histone chaperones

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146
Q

What are the 4 main mechanisms that change chromatin structure?

A

Nucleosome sliding

Nucleosome eviction

Histone variant exchange

Histone tail modification and DNA methylation

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147
Q

Sugar phosphate backbone of DNA is … charged so interactions between the histones and the DNA requires positively charged … contacts

A

Negatively
Amino acids

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148
Q

Generally, regulators bind DNA in nucleosomes with lower affinity than to naked DNA because…

A

Cis regulatory sequence facing inwards

Changes to the shape of the binding site due to associated protein binding

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149
Q

Broad promoters contain multiple
… elements and attract transcription factors that influence … in a variety of ways

A

Cis
Transcription

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150
Q

Other epigenetic mechanisms:

A

DNA methylation - direct modification of DNA bases

Interactions between DNA modification and protein modifications

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151
Q

Epigenetic’s is the study of how gene expression can be changed WITHOUT changing the DNA sequence.

What 2 mechanisms is it controlled by?

A

DNA methylation
Histone modification

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152
Q

Euchromatin is decondensed chromatin.
What causes the relaxed structure?

A

Replication
Transcription
DNA repair

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153
Q

Heterochromatin is condensed chromatin
What causes the condensed structure?

A

Inhibition of transcription
Cell division

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154
Q

What does nucleosome sliding require?

A

Histone chaperones - eg nucleosome assembly protein 1 (NAP1)

ATP dependant chromatin remodellers - eg SWI2/ SNF2

All are ATP dependant complexes which interact with histones directly

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155
Q

nucleosome eviction

A

Removal of entire nucleosome
ATP dependant process
May occur in conjunction with Histone exchange

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156
Q

What is Histone variant exchange controlled by?

A

Histone chaperones - eg Asf1

ATP dependant chromatin remodellers eg SWR complex

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157
Q

What are Histone tail modifications?

A

Changes in amino acid properties change the interaction interface with DNA

HISTONE tails help to ‘grip’ the DNA so changes here can affect the attraction between them

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158
Q

Overall charge is important for …

A

DNA: protein interactions

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159
Q

Arginine modification

A

Always retaining a + charge
Different enzymes catalyse the addition of the 1st methyl group to the addition of subsequent methyl groups
Added by Histone methyl transferases
Removed by lysine demethylases

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160
Q

Sumoylation and ubiquitinylation

A

Occurs on lysine residues
Neutralises the positive charge

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161
Q

Phosphorylation

A

Mostly at serine (but also threonine and tyrosine) residues
Generates a negative charge

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162
Q

Some modifications are in competition because:

A

Occur at the same residue - eg lysine Acetylation and methylation

Occur on consecutive amino acids, creating a steric hindrance

Occur on consecutive amino acids and binding of 1 site stimulates binding of further proteins

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163
Q

Modification by the components of the … or Coactivator determine the … of the changes

A

Mediator complex
Longevity

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164
Q

3D bending of the DNA and binding of proteins to the … may prevent transcriptional initiation or progression of the RNA pol … complex

A

Insulator
2

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165
Q

Hearing is the ability to perceive sounds.

What are sound waves?

A

Alternating high and low pressure regions travelling in the same direction
Originate from a vibrating object
Frequency of sound vibration = pitch
Amplitude = how loud (decibel, dB)

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166
Q

What is the audible range of the human ear?

A

Audible range = 20- 20 000 Hz

Hears most acutely between 500-5000Hz

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167
Q

What are the 3 main regions of the ear?

A

External ear - collects sound waves and channels them inward

Middle ear - conveys sound vibrations to the oval window

Internal ear - houses receptors for hearing and equilibrium

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168
Q

Physiology of hearing

A
  1. Auricle directs sound waves into the external auditory canal
  2. Tympanic membrane vibrates back and forth
  3. Vibrations transmit to malleus, incus and stapes
  4. Stapes vibrates in the oval window
  5. Fluid pressure waves in the peri lymph of the cochlea
  6. Pressure waves transmit from scala vestibuli to scala tympani to the round window so it bulges into middle ear
  7. Pressure waves deform walls of scalea vestibuli and scala tympani
  8. Pressure waves cause basilar membrane to vibrate
  9. Move hair cells out of spiral organ against tectorial membrane
  10. Stereocilia bend and generate nerve impulses in 1st order neurons in cochlear nerve fibres
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169
Q

Sound transduction - what happens when hair cell is at rest?

A

Stereocilia point straight up
Cation channels are partially open
Weak depolarising receptor potential
Ca+ ions enter
Low level NT release

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170
Q

Sound transduction - what happens when hair cells are stimulated?

A

Vibration of basilar membrane
Stereocilia bend and open cation channels
Larger numbers of K+ ions enter the cell
Strong depolarising potential
More Ca+ channels open
Increased NT release

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171
Q

DNA methylation

A

DNA methylation is carried out by DNA Metyl Transferases (DNMTs)

De novo DNMTs add methyl groups to unmethylated DNA - eg DNMT3a and DNMT3b

Other DNMTs add methyl groups to daughter stand during DNA replication - eg DNMT1

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172
Q

DNA methylation

A

In around 1% of nucleotides of the genome

Mostly occurs at CpG islands

Causes different effects depending on the gene sequence

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173
Q

What does DNA methylation have a role in?

A

Regulating tissue specific gene expression

Genomic imprinting

X chromosome inactivation

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174
Q

How do CpG islands affect expression?

A

Regulation of chromatin structure AND transcription factor binding by:

Less nucleosomes
Often close to TSS
Usually encompass transcription factor binding sites
Methylation of exon 1 helps recruit TF

OPEN UP STRUCTURE AND RECRUITS ENZYMES

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175
Q

How does DNA methylation affect expression?

A

Regulation of chromatin structure AND transcription factor binding by:

Recruitment of inhibitory TF
Disrupting binding to TF binding sites
Methylation of promoter causes gene silencing

STABLE SILENCING

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176
Q

What are epigenetic readers?

A

Chromatin structure can be changed with the association of a reader complex which is similar in theory to the mediator complex

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177
Q

What are epigenetic writers?

A

The writers are the enzymes that change the modifications - eg Histone acetyl transferases, Histone methyl transferases and DNA methyl transferases

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178
Q

What are epigenetic erasers?

A

The erasers are enzymes that modify or remove these marks: Histone deacetylases and lysine demethylases

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179
Q

How is epigenetics spread?

A
  1. Regulatory proteins bind to specific cis sequence
  2. These recruit Histone modifiers (writers)
  3. A reader protein recognises the modification to the Histone / DNA
  4. A writer recognises the reader and binds to it, providing a platform from which the writer can make another modification on the next nucleosome (goes back to step 3!)
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180
Q

What happens in the epigenetic modification: expression?

A

Histone tails methylation (me3)

Inhibition of DNMT

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181
Q

What is the epigenetic modification: repression?

A

Methylation of DNA and Histone tail modification

Recruit methyl binding proteins (MBD)

Recruit Histone tail modifiers (eg HDAC and HMT)

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182
Q

Barrier sequences can stop the spread of changes to chromatin structure, creating CHROMATIN DOMAINS that are regulated separately.
Examples are:

A

Tethering a chromatin domain to a large fixed site, such as the nuclear pore complex

Strong binding of a group of nucleosomes to a barrier protein

Recruitment of a mediator complex containing chromatin modifying enzymes to erase modifications that will spread changes

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183
Q

Summary of controlling gene expression

A

A) competitive DNA binding

B) masking the activation surface

C) direct interaction with the general TF

D) recruitment of chromatin remodelling complexes

E) recruitment of Histone deacetylases

F) recruitment of Histone methyl transferase

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184
Q

Epigenetic case study: Amyotrophic lateral sclerosis (ALS)

A

Progressive disease that affects motor neurons

CAUSE = overall decrease in acetylated Histone levels within motor neurones by:

Reduction in histone Acetyl transferase (HAT) activity
Increased HDAC activity

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185
Q

Epigenetic’s case study: Fragile X

A

Caused by silencing of FRM1 gene

CGG tri nucleotide repeats in the 5 UTR FRM1 associated with disease onset

Expansion of number of repeats (>200) - hyper methylation of promoter in an attempt to turn off expression

C turned into 5mC by DNMTs = interaction with Histone marks / compacted chromatin structure / harder for TF to be recruited and enhance expression

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186
Q

What is cancer?

A

Defined as the continuous uncontrolled growth of cells

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187
Q

What are tumours?

A

Any abnormal proliferation of cells
They are classified as to their cell type
Can arise from any cell type in the body

Benign - stay confined to original location
Malignant - capable of invading surrounding tissue and spreading to entire body

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188
Q

Epithelial cells can give rise to tumours called…

A

Carcinomas

Carcinomas cause 80% of cancer related death

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189
Q

Examples of carcinogens that can cause cancer

A

Ionising radiation - x rays, UV light

Chemicals - tar from cigarettes

Virus infection - papilloma virus can be responsible for cervical cancer. Hepatitis B and C viruses = liver cancer

Hereditary predisposition - some families are more susceptible to getting certain cancers

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190
Q

What 7 type of cancers can alcohol cause?

A

Mouth
Upper throat
Oesophagus
Breast
Liver
Bowel

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191
Q

4 ways that alcohol causes cancer

A

Damages cells
Increases damage from tobacco
Affects hormones linked to breast cancer
Breaks down into cancer causing chemicals

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192
Q

Tobacco can cause … types of cancer

A

14

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193
Q

Chemical carcinogens

A

Thousands have been identified
Broad range of chemical structure
Classified as direct acting and indirect acting

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194
Q

Carcinogens cause mutations:

A

Can cause DNA damage, change sequence of DNA bases, change codon and affect protein function

Most commonly occurring spontaneous change in DNA:
Oxidative damage
Spontaneous purine hydrolysis
Deamination

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195
Q

How to identify carcinogens (compounds that can cause mutations): Ames Test

Tris 2,3- dibromopropyl phosphate used as a … in plastic and textiles. Furylfuramide used as an … in food in Japan
Using bacteria that require … to grow > growth means they attained the ability to … histidine as a result of mutation (revertant bacteria)

A

Flame retardant
Antibacterial additive
Histidine
Synthesise

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196
Q

Benz(a)pyrene

Epidemiological studies suggested smokers had …incidents of lung cancer

Cigarette smoke contains about … carcinogens and they can induce mutations in different genes

60% of lung cancers have inactivating …in the p53 gene, which is a major tumour … gene

A

High
60
Mutations
Suppressor

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197
Q

Purine hydrolysis

A

DNA damage events in a single cell range from 10^4 to 10^6

Under physiological conditions, spontaneous purine hydrolysis takes place leaving a sugar without the attached base leading to generation of AP site

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198
Q

Routes to oxidative DNA damage dependant mutagenesis

A

Mispairing of 8 oxoG with adenine during replication leads to C to A causing GC base pair to mutate to TA

GC to TA mutation can also result from replication encountering an AP site

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199
Q

Deamination can induce point mutations and mismatch

A

Cytosine and 5 methylcytosine are most common deamination reactions

DNA bases can be deaminated and if unrepaired can cause mutations

Deamination can change a GC base pair to AT

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200
Q

Adenine and guanine can also be damaged by …
Both hypoxanthine and Xanthine can pair up with …

A

Deamination
Cytosine

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201
Q

Deamination changes GC base pair to … base pair

A

AT

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202
Q

Incidents of cancer increase with…

A

Age

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203
Q

Multi hit model of cancer induction explains why cancer incidence rise with age:

A

Predicts increase in cancer incidence with age

Cancers arise by an evolutionary selection process following the theory of ‘survival of the fittest’

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204
Q

What are the 4 mutations of the Multi hit model of cancer induction?

A

1st mutation - gives a slight growth advantage to mutant cells

2nd mutation - mutant cells grow more uncontrollably and form a small benign tumour

3rd mutation - mutant cells overcome constraints imposed by tumour surroundings. Outgrow others to form a mass of cells, each of which has all 3 genetic changes: 1st, 2nd and 3rd mutations

4th mutation - cells can escape (and survive) into the bloodstream and establish daughter colonies at other sites (hallmark of metastatic cancer)

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205
Q

Exogenous and endogenous agents can …the DNA

A

Damage

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206
Q

Carcinogens are mutagens, they induce changes in DNA sequence
IF REMAIN UNCORRECTED can lead to …

A

Mutations

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207
Q

Ames test can be used to detect potential …

A

Carcinogens

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208
Q

Repeated mutation and their accumulation over time in cells explains why tumour formation is a gradual … process which can take several years for cancer to develop

A

Multi step

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209
Q

Pentose phosphate backbone

A

5’ phosphate
3’ hydroxyl
5-3 directionality
Phosphodiester bond
Bases on the same side
Negative charge outside
Sequence of bases forms the primary structure
3 hydroxyl is the substrate for DNA polymerase

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210
Q

SSBs are constantly and spontaneously … in cells

A

Generated

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211
Q

SSB repair is promoted by Poly ADP ribose polymerase (PARP) enzyme.
This leads to … of one of the EXCISION REPAIR pathways to … the damaged DNA

A

Activation
Repair

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212
Q

Inability to repair SSB converts them into DNA double strand break (DSB) when the cells …

A

Divide

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213
Q

DSB are highly … to cells, any unresolved DSBs are sufficient to kill cells: this is what … causes

A

Toxic
Radiotherapy

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214
Q

DNA exonuclease can hydrolyse a phosphodiester bond on DNA …

A

Terminini

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215
Q

DNA endonuclease creates a nick in between DNA chain by … (hydrolysis) a phosphodiester bond

A

Cutting

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216
Q

DNA ligase closes the nick by forming a … bond

A

Phosphodiester

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217
Q

DNA polymerases mediated proof reading

A

DNA pol is the first line of defence against mutations

DNA pol in E coli introduces 1 wrong base for every 10K bases incorporated, but the mutation rate is 1 wrong base per 1 billion

Low mutation rate is due to the proof reading (3-5 exonuclease) function

Proof reading is vital for all cells to avoid excessive mutations

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218
Q

PARP1 is critical in recruiting DNA repair proteins

A

PARP identifies DNA damage and signals the need for repair

PARP1 detects DNA damage via its DNA binding domain

Activated PARP1 adds ADP ribose units to PARP1 and leads to the formation of long and branched chains of poly (ADP ribose) (PAR)

These PAR chains create a scaffold that recruits critical proteins for DNA repair

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219
Q

Replication errors are corrected by mismatch excision repair (MER)

A

Inheritable loss of function mutation in msh2 or mlh1 genes cause predisposition to non polyposis colorectal cancer

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220
Q

What is DNA pol b?

A

A specialised DNA polymerase used in repair

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221
Q

Nucleotide excision repair (NER) corrects … DNA damage

A

UV induced

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222
Q

NER defects lead to …

A

Xeroderma pigmentosum

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223
Q

Repair of DNA double stand break (DSB) by homologous recombination

A

DNA DSBs are highly toxic, a few sustained DSBs are enough to kill a cell

Radiotherapy and many anti cancer drugs destroy tumour by causing DNA double stand breaks

Incorrect joining of DSBs can create hybrid genes and can place a low expression gene under control of a strong promoter

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224
Q

Cells use 2 processes to carry out DSBs repair:

A
  1. Non homologous end joining (NHEJ) - error prone
  2. Homologous recombination (HR)
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225
Q

What is synthetic lethality (SL)?

A

Exploiting tumour defects to cure cancer

It arises when a combination of deficiencies in the expression of two or more genes leads to cell death, whereas a deficiency in only one of these genes does not

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226
Q

PARP repairs DNA SSBs so they are … converted into double strand breaks

PARP inhibitor drugs cause … to convert to DSBs

A

Not
SSB

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227
Q

DNA damage is constantly occurring and therefore DNA repair pathways evolved to maintain …
Their failure can lead to …

A

Genomic integrity
Diseases

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228
Q

At least 3 types of excision repair mechanism can correct SS DNA damage in the DNA using … sets of enzymes in a sequential manner

A

Specific

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229
Q

PARP (by regulating SSB repair) and BRCA (by regulating DSB repair) coordinate in maintaining the … of the DNA in …

A

Integrity
Breast cancer

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230
Q

BRCA (in breast / AR in prostate) deficient tumours cannot afford to repair DSB, they rely on … activity to ensure DNA SSB are repaired and DO NOT progress to …

A

PARP
DSB

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231
Q

BRCA and PARP are synthetically lethal in … cancer.
Androgen receptor AR and PARP are synthetically lethal in … cancer

A

Breast

Prostate

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232
Q

Hallmarks of cancer are biological capabilities acquired during multistep development of human tumours

What are they?

A
  1. Sustaining proliferative signalling - oncogenes
  2. Evading growth suppression - tumour suppressor genes
  3. Reprogramming of energy metabolism
  4. Inducing angiogenesis
  5. Genome instability and mutations
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233
Q

What are the 3 broad categories of genes implicated in cancer?

A

Proto oncogenes

Tumor suppressor genes

Genome maintenance genes - mutation allows propagation of gene mutations as the DNA repair is inefficient

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234
Q

Proto oncogenes and oncogenes

A

Rationale for their existence: required for normal growth and proliferation

Proto oncogenes (ras) - gain of function mutations convert them into oncogenes

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235
Q

Activation of oncogenes can trigger …

A

Cancer

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236
Q

Mechanisms of gain of function mutation

A
  1. Point mutation (a change in single base pair) - altered protein product
  2. Chromosomal translocation - fuses 2 genes together to produce a hybrid gene
  3. Amplification - generation of numerous proto oncogene copies, leading to overproduction of the encoded protein
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237
Q

Loss of ligand (growth factor) dependant receptor activation

A

As a result of ‘gain of function point mutations’ in the receptor tyrosine kinases, the requirement of growth factor (ligand) to trigger receptor dimerisation may be abolished allowing cells to grow rapidly and uncontrollably

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238
Q

Oncogenic receptor can promote proliferation without …

A

Growth factors

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239
Q

Growth factor can trigger autocrine activation in cancer

A

Normally the receptor and ligand are produced by different cells

In some cancers, BOTH of these can be made by the same cell, losing regulated control of growth / division
Eg EGF and EGFR being made by the same cells in cancer

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240
Q

Her 2 kinase is an oncogene

A

1/3 Breast tumours express Her2, triggering sustained proliferation of cancer

Trastuzumab (a monoclonal antibody) can target Her2 leading to repression of 1) Her2 mediated growth signalling
2) destruction by the immune system

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241
Q

Transcription factors can drive cancers

A

Prostate cancer stimulated by androgen (testosterone), the male sexual hormone > activates androgen receptor AR

Breast cancer stimulated by estorgens (female sex hormones) > activates estrogen receptor ER

AR and ER are example of nuclear hormone receptors

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242
Q

Nuclear hormone receptors are therapeutic targets

A

Enzalmutamide = AR antagonist

Tamoxifen = ER antagonist

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243
Q

Tumour suppressor genes

A

Ensure cells with oncogenes are repaired / killed, hence anti cancer

As long as they are intact, cancer cells should NOT survive

Many cancers have inactivation of tumour suppressors and that’s how cells with oncogenes can make tumours

Rationale for existence: controlling cell cycle checkpoints and development. They also regulate ‘apoptosis’

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244
Q

Tumour suppressor genes

Eg Rb / APC / p53

A

Loss of function mutation deletes an important brake on the cell cycle or checkpoint control

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245
Q

Hypoxia =

A

Lack of oxygen

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246
Q

Angiogenesis =

A

Formation of new blood vessels

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247
Q

Apoptosis =

A

Cell death

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248
Q

Senescence =

A

Cellular ageing

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249
Q

Normal cells

A

Proto oncogenes + tumour suppressor genes =
Regulated cell growth and proliferation

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250
Q

Cancer cells

A

Gain of function mutation in proto oncogene + loss of function mutation in tumour suppressor genes =
Abnormal growth and cancer

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251
Q

Hallmarks are …

A

Specific characteristics found in cancer cells

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252
Q

Oncogenes promote … while tumour suppressor genes repress ..

A

Tumour growth
Cancer

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253
Q

Nuclear receptors as well as many gene fusions can become … as a result of mutations

A

Oncogenes

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254
Q

AR in … and ER in .. are potential oncogenes

A

Prostate
Breast

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255
Q

One of the ways that ATM participates in DNA repair is via activation of … tumour suppressor function

A

P53

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256
Q

Rb protein puts a break in … and thus cell division by trapping E2F transcription factors

A

DNA replication

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257
Q

Viruses not only carry oncogenes but some of their proteins can … tumour suppressor gene function

A

Inactivate

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258
Q

Blocking oncogene function forms the basis of …

A

Cancer therapy

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259
Q

Tumour suppressor genes maintain … and ensure cells don’t divide with a damaged DNA

A

Genomic integrity

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260
Q

What are the effects of condensed chromatin structure? (Making heterochromatin)

A

Distance between nucleosomes decreases

Further packaging of DNA into solenoid then looped structures, which requires interaction with another level of proteins

Cellular effects: decrease in transcription from some gene promoters

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261
Q

What can we monitor if we want to determine the state of the chromatin structure?

What can we monitor to identify the effect that it’s changes in structure are having?

A
  1. mRNA levels
  2. Measure modification status of Histone complex - if acetylated lysine on histone H3 = transcription about to be turned on. If methylated = transcription turned off
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262
Q

What are the steps of Chromatin Immuno Precipitation (CHIP)?

A
  1. Cross link DNA and proteins and isolate chromatin
  2. Sonicate or digest chromatin
  3. Immuno precipitate, reverse cross linking, purify DNA
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263
Q

What comes after CHIP?

A

Detect by microscopy (chip, chip)
Or

Detect by sequencing (chip seq)
Or

Western blotting procedure

264
Q

Methods of detecting DNA methylation:
BS Seq (bisulphite sequencing)

A
  1. Sonicate DNA
  2. Bisulphite conversion
  3. PCR amplification: Sanger sequencing or pyro sequencing (faster)
265
Q

Sanger (dideoxy) sequencing is based on…

A

DNA replication

266
Q

Pyro sequencing is based on…

A

PCR

267
Q

Viruses not only carry … but they can cause … by inhibiting tumour suppressor gene function

A

Oncogenes
Cancer

268
Q

In normal cells, oxygen determines the destiny of …

Lactate is formed under conditions of …

A

Pyruvate

Hypoxia

269
Q

Warburg effect

(Aerobic glycolysis)

A

Warburg observed that tumours were taking up enormous amounts of glucose compared to surrounding tissue

This glucose was fermented to produce lactate even in the presence of oxygen

Because aerobic glycolysis is energetically INefficient - cancer cells increase glucose uptake

270
Q

2 deoxyglucose phosphate (2 DG P) competitively inhibit the production of …

A

Fructose 6 phosphate

271
Q

2 hydroxyl glutarate (2HG) is an Oncometabolite

A

Mutant IDH (common in cancers) converts iso citrate into 2HG

2HG inhibits several enzymes that require alpha keto glutarate

272
Q

Benign tumours

A

DO NOT invade or metastasise eg Wart

273
Q

Malignant tumours

A

Can invade nearby tissue, spread and seed additional secondary tumours in distant organs

274
Q

VEGF promotes angiogenesis.

What is this?

What stimulates VEGF production?

A

Formation of new blood vessels to allow nutrition to reach tumour

Stimulated by low oxygen conditions - hypoxia

275
Q

How cells sense and adapt to oxygen availability

A
  1. Low oxygen conditions enhance erythropoietin (EPO) levels
  2. EPO is a growth hormone produced by the kidney
  3. Under normoxia (normal blood oxygen concentration) EPO is synthesised by inner cortex
  4. Under hypoxia, cells of kidney can produce more than 100x EPO over normoxia
  5. Tumour hypoxia is a COMMON phenomenon in solid tumours
276
Q

Erythropoietin (EPO) stimulates red blood cell …

A

Production

277
Q

Hypoxia induces HIF 1a transcription factor activation that promotes aggressive … phenotype

A

Tumour

278
Q

Pathways regulated by hypoxia:

A

Angiogenesis, tissue invasion and metastasis

279
Q

Expression of VEGF is responsible for induction of … which is associated with tumour …

A

Angiogenesis
Progression

280
Q

Altered bioenergetics in cancer cells is energy … but makes cancer cells fitter and more …

A

Inefficient
Competitive

281
Q

Oncometabolites can be produced as a result of … in a metabolic pathway

A

Mutations

282
Q

Cancer cells make new highways by … to spread themselves in distant organs

A

Angiogenesis

283
Q

HIF 1a promotes angiogenesis by activating genes such as … that promote angiogenesis in cancer

A

VEGF

284
Q

What is the central dogma?

A

Genomic DNA > transcription > mRNA > translation > protein

285
Q

Where are prokaryote 70S ribosomes (30 +50) found?

A

Free in the cell

286
Q

Where are eukaryote 80S ribosomes (40+60)found?

A

In the cytoplasm, ER associated

Can be localised to specific intracellular areas eg dendrites in neuron

287
Q

What is the initiation step?

A

Recruitment of initiation factors and ribosomal subunits

Recognition of AUG start codon

288
Q

Why is the initiation step the target for control mechanisms?

A

Because it is the rate limiting step of mRNA translation

289
Q

What is the elongation step?

A

Recruitment of elongation factors and acyl tRNAs

Building of polypeptide chain

290
Q

What is the termination step?

A

Recruitment of release factors

Recognition of stop codon and release of polypeptide chain

291
Q

How is eukaryotic translation different to prokaryotic translation?

A

Most of it is cap dependant - starts at 5 prime cap (ribosome had to find the beginning)

In prokaryotes, a Shine Dalgarno sequence helps to recruit ribosomes directly to RNA at any point of that mRNA

292
Q

What is the function of the 5 cap structure?

A

Protective for degradation and crucial for translation

293
Q

What is the open reading frame ORF?

A

Only part that contributes to the code of the protein being produced

294
Q

What is the function of the 3 poly A tail?

A

Translational control

295
Q

What happens during translation initiation?

A

Cap binding

5-3 scanning to find AUG, requires: unfolding of 5 UTR and ATP hydrolysis

AUG recognition - codon / anti codon pairing

GTP hydrolysis / tRNA delivery

eIF release + large subunit 80S joining

GTP hydrolysis x2 and eIF release

296
Q

Why is it important to locate the right AUG start codon?

A

To avoid frame shifts

Particularly important for 1st codon to match up the right AA in the right place

Nothing else will be coded for properly for the remainder of translation otherwise

297
Q

Prokaryotic and eukaryotic translation have similar pathways, especially in the … phase

A

Elongation

298
Q

Rate limiting steps are key targets for … and …

A

Regulation

Dysregulation

299
Q

What is involved in cap recognition?

A

eIF4E sequestration, phosphorylation

eIF4G cleavage

300
Q

What is involved in ternary complex formation?

A

eIF2 - GDP recycling

eIF2 phosphorylation

301
Q

Initiation is the most … step

A

Regulated

302
Q

What are the key steps during translation initiation?

A

Cap recognition

Scanning

AUG start codon recognition

303
Q

What happens to eIF4G structure in apoptosing cells?

A

Caspase 3/7 cleavage of eIF4G

Disruption of cap binding complex

Cap dependant translation

Overall down regulation of translation in apoptosing cells

304
Q

What is an IRES?

A

A structured RNA domain that recruits the ribosome

305
Q

IRES dependant translation

A

No need for the cap recognition and scanning

Require only few eIFs

Present on cellular mRNAs and viral RNAs

306
Q

What is a cistron?

A

Open reading frame that encodes for reporter proteins

307
Q

What normally happens with cistrons in mRNA?

A

If 2 open reading frames are next to eachother in capped mRNA, the 1st cistron will be translated to = product

But at the end of translation, the ribosome will terminate so no translation will happen downstream of 2nd cistron

308
Q

How can we test whether RNA sequence is functioning as an IRES?

A

Put the sequence between two cistrons!!

309
Q

What are bicistronic reporters ?

A

Gold standard to define IRES function

310
Q

Unstructured 5 UTR =

A

Easy scanning

311
Q

Structured 5 UTR =

A

Tough scanning - poorly translated in a cap dependant environment

312
Q

How do eIF4E levels regulate cap recognition?

A

eIF4E over expression increases efficiency of cap binding

313
Q

mRNAs with structured UTR include:

A

c myc

VEGF

cyclin D1

FGF

314
Q

How does increased eIF4E levels lead to cancer?

A

Leads to increased translation of a subset of normally poorly translated mRNAs - eg VEGF, c myc

Causes angiogenesis, proliferation, evasion of apoptosis, metastasis

= CANCER

315
Q

Breast / prostate cancers display high levels of…

A

Phospho 4E BP

316
Q

Examples of mitogenic signals?

A

Cytokines

Hormones

Growth factors

317
Q

Regulation of cap recognition

A

eIF4A /B phosphorylation, eIF4E over expression, 4E BP phosphorylation and eIF43 phosphorylation all lead to..

Increased cap recognition which leads to …

Increased translation of specific mRNAs

318
Q

eIF2 GDP means translation is…

A

OFF

319
Q

eIF2 GTP tRNA means translation is…

A

On

320
Q

How is ternary complex formation regulated?

A

Regulated by phosphorylation of eIF2 alpha subunit of eIF2

321
Q

dsRNA binding motifs are linked to…

A

Viral RNA recognition

322
Q

Kinase (ser / thr) catalytic domain is linked to …

A

eIF2 phosphorylation

323
Q

PERK (PKR like ER localised kinase) prevents from protein … related to ER stress.

A

Overload

324
Q

PERK defect can lead to Wolcott Rallison syndrome.
What is this?

A

Growth retardation and diabetes

Imbalance between folded and unfolded protein

325
Q

What happens when there is a eIF2B mutation?

A

Impaired eIF2 recycling = less Protein synthesis and fail to recover protein synthesis after stress

326
Q

eIF2B mutations cause disease such as leukodystrophy (vanishing white matter)
What is rhis?

A

Neurological deterioration

Motor dysfunction

327
Q

eIF4E has a key role in translation …

A

Regulation

328
Q

eIF2 recycling is regulated via …

A

eIF2 alpha phosphorylation

329
Q

eIF2 alpha phosphorylation leads to global … in translation

A

Reduction

330
Q

What happens when things go right in translation?

A

Translation initiation proceeds normally

Multiple ribosomes (poly somes) translate nascent peptide chains

331
Q

What happens when there’s a PTC (premature termination codon) in translation?

A

mRNA incorrect > NMD nonsense mediated decay > mRNA decay to avoid synthesis of non functional or potentially toxic truncated proteins

332
Q

What are PTCs and what causes them?

A

Stop codon further upstream than we would expect

Could get introduced via transcription error or a mutation in DNA

333
Q

What happens during nonsense mediated decay NMD?

A

mRNA export

Monosome stalled onto PTC

Stalled ribosome and EJC recruit the SURF complex triggering NMD

Truncated polypeptides are ubiquitnated and degraded by proteasome

334
Q

What are the quality control pathways?

A

Nonsense mediated decay NMD - premature termination codons

No go decay NGD - stalled in translation mRNAs

Non stop decay NSD - mRNAs without natural stop codons

335
Q

Most mRNA regulation occurs though the … and … UTRs

A

5
3

336
Q

What are the elements of the 5 prime UTR?

A

IRES
5 UTR structures
Recruitment of RNA binding proteins - eg IRP
Upstream ORF - uORF

337
Q

What are the elements of the 3 prime UTR?

A

Recruitment of RNA binding protein

miRNA binding site

338
Q

How does an upstream ORF uORF repress translation?

A

Ribosomes dissociate after translating the uORF

Ribosomes stalled by the uORF encoded peptide

339
Q

What happens under normal translational conditions?

A

Ternary complex abundant

GCN4 not produced

340
Q

What is GCN4?

A

A transcription factor activating more than 50 genes involved in amino acid synthesis

341
Q

What happens under amino acid stress conditions in translation?

A

Ternary complex limiting, only authentic AUG recognised

uORF translation impaired by eIF2 alpha P

GCN4 translation promoted

342
Q

Caudal mRNA

A

Caudal is an important patterning molecule during embryogenesis

4E HP blocks eIF4F binding and the translation of caudal mRNA

343
Q

Bicoid

A

Bicoid (RBP) interacts with the 3UTR of caudal

Bicoid recruits 4E HPn(4E homologous protein) to caudal cap

344
Q

msl2 mRNA

A

Is translated in males but not female during development

345
Q

SXL - sex lethal

A

And UNR are RBPs that repress msl2 translation by binding the 3 UTR

Prevents 43S binding by locking 5-3 interactions

SXL only expressed in females

346
Q

What is the epi transcript ome?

A

Where RNA bases can be chemically modified

347
Q

m6A

A

Promotes RNA translation by multiple mechanisms

Reader proteins or METTL3 can bind m6A and promote cap dependant translation

Translation factor eIF3 can be recruited by mRNA directly

348
Q

M6A controls multiple reversible RNA … processes

A

Regulatory

349
Q

5 UTR and 3 UTR contain a variety of regulatory elements that mediate translational ..

A

Control

350
Q

RNA binding proteins are key to translational …

A

Regulation

351
Q

Translational control impacts many areas of biology including:

A

Stress response
Early development
Sex determination

352
Q

What do tRNAs do?

A

Decode the mRNA sequence during protein synthesis at the ribosome

353
Q

Ribosomal RNA is the most abundant RNA in the cell!

A

80% of total RNA

Transcribed by Pol 1 - expect for 5S by pol 3

354
Q

What are the two eukaryotic ribosome subunits?

A

40S - 18S rRNA

60S - 5S, 5.8S, 28S

355
Q

What are the 5.8, 18 and 28S subunits made from?

A

Single transcript - the 45S precursor
= rRNA processing in the nucleolus

356
Q

What are snRNAs?

A

Small nuclear RNAs

U rich sequences involved in the splicing of pre mRNA (U1 2, 4, 5, 6)

Highly expressed and evolutionary conserved

357
Q

What are small nucleolar RNAs snoRNAs?

A

Help to process and chemically modify rRNAs

Main function in nucleolus > RNA processing

Encoded with introns of Pol 11 transcribed genes

358
Q

What RNA modifications do snoRNAs guide?

A

C/D box - directs 2’ O ribose methylation by recruiting a methyl transferase enzyme

H/ACA box - recruits an enzyme that converts uridine to pseudouridine

359
Q

Current data suggest that …. Human miRNAs target about 45 000 miRNA sites in about … of human protein coding genes, mainly in 3’ UTRs

A

5000
60%

360
Q

What are microRNAs and small interfering RNAs?
miRNAs and siRNAs

A

Specifically bind to complementary sequences located in 3 UTR regions of mRNAs

361
Q

How do miRNAs and siRNAs affect mRNA and translation?

A

miRNAs - promote deadenylation, translation repression and decay

siRNAs - cleavage of mRNA and exosome mediated degradation

362
Q

Micro RNAd repress translation by … hybridisation with target mRNAs in the cytoplasm

A

Imperfect

363
Q

What are the seed sequence miRNA nucleotides that are critical for targeting specific sequences?

A

2-7

364
Q

Small interfering RNAs cleave mRNAs upon perfect hybridisation

A

Defence mechanism against invading double stranded RNA viruses and transposable elements

Crucial in plants, worms and insects

Less in mammals

365
Q

What does stable dicer gene knock out do?

A

Eliminates generation of miRNAs in mammals and is embryonic lethal

366
Q

What does conditional dicer knockout do?

A

Leads to defects in tissue morphogenesis / development

367
Q

miRNAs and disease

A

Control genes with crucial functions in cell proliferation, development, inflammation and ageing

Have been linked to cancer oncogenes and tumour suppressors

Target for bio markers and drug development

368
Q

Long NON coding RNAs (IncRNAs)

A

Transcribed by pol 11

Contain 5’ cap and poly A tail at 3’ end

Not translated into proteins

Tissue / cell type specific expression

Involved in cell differentiation, development and anti viral responses

Many functions are unknown!

369
Q

LncRNAs can interact with … to execute … functions in nucleus or cytoplasm

A

Proteins, RNA and DNA
Regulatory

370
Q

Nuclear LncRNAs control chromatin structure / transcription in … or …

A

Cis
Trans

371
Q

What is X inactivation specific transcript XIST?

A

A large 17kb cis acting regulatory LncRNA

Associates with X chromosome

Initiates histone modifications which results in heterochromatin formation

372
Q

Cytoplasmic lncRNAs have diverse functions including:

A

microRNA decay
RBP decay
Protein turnover
Organelle function
Signalling
mRNA translation and degradation

373
Q

How do lncRNAs regulate mRNA stability?

A

The lncRNA TINCER interacts with complementary sequences and recruits RNA binding protein STAU1

THIS promotes stability of mRNA

374
Q

How do lncRNAs regulate mRNA translation?

A

Under stress conditions, lncRNA antisense moves from nucleus to cytoplasm and binds to end of Uchl1 mRNA to promote translation

375
Q

LncRNAs like NORAD can act as a … for RNA binding proteins and can control cell …

A

Decoy
Mitosis

376
Q

What does NORAD +/+ mean?

A

Normal PUMILIO activity

Normal mitosis

377
Q

What does NORAD -/- mean?

A

Hyperactive PULMILIO

Aberrant mitosis

378
Q

Circular RNAs circRNAd can acts as decoys for miRNAs and RBPS

A

Generated via back splicing mechanism

Highly abundant and stable

379
Q

What are the most abundant non coding RNAs?

A

tRNAs and rRNAs

380
Q

What do miRNAs and siRNAs conteol?

A

Gene expression post transcription ally by annealing to sequences in 3’ UTRs of mRNA targets

381
Q

What does processing of miRNAs / siRNAs involve?

A

Drosha, Dicer to form the RISC complex

382
Q

What is the function of lncRNAs in eukaryotes?

A

Nuclear and cytoplasmic functions in gene expression and control

383
Q

Does RNA processing happen in prokaryotes?

A

No!

384
Q

In eukaryotes, only … of all RNA in the cell is mRNAs!

A

1.5%

385
Q

Pre mRNA processing involves what?

A

Pre mRNAs are capped, polyadenylated and spliced in the nucleus

386
Q

How are RNA processing factors recruited to pre mRNAs?

A

They are recruited co - transcriptionally

By carboxy terminal domain CTD of pol 11

387
Q

Where is the 7 methyl guanosine cap added?
And what does it do?

A

Added at the 5’ end of eukaryotic pre mRNAs

Protects the mRNA from degradation by nulceases

388
Q

What does the Spliceosome do?

A

Catalyses 2 transesterification reactions that joins 2 exons and REMOVES the intron as a Lariat structure

389
Q

What do the network of interactions between snRNPs and splicing factors determine?

A

Splice site selection

390
Q

What do RNA binding proteins (RBP) do?

A

Bind to specific sequences near the splice site

Regulate alternative splicing (SR proteins > activators > hnRNPs > repressors)

391
Q

What does cleavage and polyadenylation at the 3’end of pre mRNAs involve?

A

Diverse RBPs and complexes

Alternative polyadenylation can have important physiological implications (eg antibodies)

392
Q

What does an mRNP exporter ensure?

A

Directional export of mRNAs from nucleus to the cytoplasm through the NPC

The mRNP exporters are phosphorylated in the cytoplasm and release the mRNA cargo

393
Q

RNA processing starts…

A

Co transcriptionally

394
Q

The 5’ cap marks RNA molecules as mRNA
What is its function?

A

Regulates nuclear export of mRNAs

Protects mRNAs from RNA digesting enzymes (5’ exoribonucleases)

Promotes translation through interaction with a translation initiation factor (eIF4E - the cap binding protein)

395
Q

What is splicing?

A

The removal of introns from the pre mRNA

396
Q

What do consensus sequences do?

A

Define the splice sites in eukaryotic pre mRNAs

397
Q

What is the spliceosome made up of?

A

5 small nuclear RNAs - snRNAs
U1, U2, U4, U5 and U6 between 107-210nts long

170 proteins

398
Q

Exons are generally .. times shorter and more uniform than introns

Since introns can be very long, additional strategies are required to improve … selection

A

10

Splice site

399
Q

What is alternative splicing in eukaryotes?

A

Generating mRNA variants from the SAME gene

400
Q

What is the most extreme example of alternative splicing?

A

The Drosophilia DSCAM gene

401
Q

What are the patterns of alternative splicing?

A

Exon skipping - most prominent in eukaryotes

Intron retention

Alternative 5’ and 3’ splice site

Mutually exclusive exons

402
Q

What diseases can mutation of splice sites or splicing factors cause?

A

B-thalassemia - autosomal recessive blood disorder. Mutation of splice site in B globin gene

Myotonic dystrophy - neuromuscular disease. Depletion of MBNL splicing factor

Cystic fibrosis

Parkinson’s disease

Premature ageing

Cancer

403
Q

What is the 3’end of eukaryotic mRNAs determined by?

A

By the processing of pre mRNA

404
Q

What is the 50-250 nts of adenosine added of the 3’ end of the last exon of eukaryotic mRNAs called?

A

The poly A tail

405
Q

Cleavage requires a protein complex.
What does it consist of?

A

CPSF

CstF

TWO cleavage factors

Poly A polymerase - PAP

406
Q

What is the function of the poly A tail in eukaryotes?

A

Required for export of mRNA from the nucleus to the cytoplasm = binding of PABPII

Promotes translation initiation and translation

Stabilises the mRNA > shortening of polyA tail may lead to reduced translation and eventual decay of the mRNA!

407
Q

How does the processed mRNA get out of the nucleus?

A

Small molecules and proteins (<60 kDa) can diffuse through membrane

Macromolecular complexes (eg RNPs) need active transport

408
Q

What does the limited correlation between mRNA and protein levels in the cell provide evidence for?

A

Post transcriptional control at the global level

409
Q

Many mRNAs are transported to specific sub cellular locations by what?

A

RNA binding proteins - that bind to particular elements within the mRNAs (often reside within the 3’ UTRs)

410
Q

What can translation be controlled globally by?

A

Initiation factors

More specifically by RNA binding proteins and non coding RNAs
Eg IRP - control of intracellular iron concentrations

411
Q

mRNAs are preferentially degraded in what?
What is deadenylation followed by?

A

Processing bodies (P bodies)

Followed by activity of decapping enzymes and exonucleolytic degradation

412
Q

What is the problem with post transcriptional control by RNA binding proteins and non coding RNAs?

A

Gene activity / mRNA levels do NOT necessarily correlate with protein abundance

413
Q

What are the possible fates of an mRNA molecule in the cytoplasm?

A

Translation (ribosome)
Localisation (organelle)
Storage (granule)
Decay (exo / endo nuclease in P bodies)

414
Q

What are the reasons for RNA localisation?

A

Target protein to appropriate region in cell

Prevents expression elsewhere

Response to local requirement (NT production in neurons)

Independent control in different cellar
Regions

Localised synthesis necessary for assembly of protein complexes

More efficient transport (one mRNA molecule vs many proteins)

415
Q

RNA localisation is … and occurs in … as well!

A

Universal

Prokaryotes and archea

416
Q

What are the molecular mechanisms for mRNA localisation? + examples

A

Directed transport on cytoskeleton - eg neuronal RNPS along microtubuli, ASH1 mRNA bud yeast tip

Random diffusion and trapping - eg drosophilia mRNAs enriched in pole plasma

Generalised degradation in combination with local protection by trapping - same as above

417
Q

Cis acting elements in the mRNA provide the … for mRNA localisation

A

Code

418
Q

What is the ASH1 protein?

A

It is a transcriptional repressor of HO = No HO transcription and No switching

HO expression is required for mating type switching

419
Q

How is mRNA delivered to the bud tip?

A

She2, she3 and myosin4 proteins form a complex with ASH1 mRNA for transport to the bud tip of the daughter cell

420
Q

What is Khd1p?

A

A translational repressor that prevents translation of ASH1 mRNAs during transport to the bud tip

421
Q

How do we visualise localised mRNAs in vivo?

A

U1A GFP fusion protein is tethered to the mRNA via U1A binding sites > this enables visualisation of mRNA via GFP

422
Q

What are the 2 major modes of translational regulation?

A

Global regulation - modification of translation initiation factors or their regulators

MRNA specific regulation - RNA structure / specific RBPs / microRNAs

423
Q

How do iron regulatory proteins prevent recruitment of small ribosomal subunit?

A

Bind to iron response element IRE and prevents recruitment of the 43S pre initiation complex via Steric Hindrance

424
Q

What happens in iron starvation post transcriptional control?

A

NO ferritin made - ferritin > storage (translation blocked)

Transferrin receptor made - transferrin > iron uptake!
(mRNA stable and translated)

425
Q

What happens in excess iron post transcriptional control?

A

Ferritin made - mRNA translated

No transferrin receptor made - mRNA degraded

426
Q

How does degradation of eukaryotic mRNAs work?

A

Shortening of poly A tail to <20 bases = stops translation

Degradation by exonucleases

427
Q

What are P bodies?

Where are they especially visible?

A

Condensed aggregates of mRNAs and proteins in the cytoplasm

In stressed cells - leading to rapid shut down of translation

428
Q

Anatomy of a prokaryotic gene

A

Enhancer
Promoter
Transcription start site

429
Q

Anatomy of a eukaryotic gene

A

Enhancer
Promoter
TSS
5’ UTR
start codon
Exons
Introns
Donor, acceptor and splice sites
STOP codon
3’ UTR
Polyadenylation site

430
Q

What are levels of protein generated controlled by?

A

Rate of transcription
Rate of mRNA degradation
Rate of protein synthesis
Rate of protein degradation

431
Q

What does it mean if a small amount of RNA is transcribed?
How will this affect translation?

A

Slow rate of transcription or a high degradation rate

Small amount of protein will be translated

432
Q

What is the promoter?

A

Non coding part of the gene that controls where transcription starts and which direction and what strand it occurs on!

433
Q

What does the promoter contain?

A

-10 and -35 consensus sequences, TSS that recruit proteins able to assist in the control of initiation of transcription

434
Q

What governs protein - DNA interactions?

A

DNA recognition sequence
Protein structure - can it bind as a monomer / dimer etc
Mediator contents
DNA packaging

435
Q

What does DNA recognition depend on?

A

Protein conformation and DNA structure

436
Q

Why must the protein interface match the DNA shape?

A

Enables electrostatic interactions
Fine tunes recognition of a specific sequence
Changes to shape of DNA or protein can affect the strength of binding - stronger the interaction, the more it will stimulate transcription! Needs to be a nice stable interaction

437
Q

What are the types of DNA binding proteins?

A

Homeodomain containing proteins - eg HOX
Beta sheet recognition protein -eg p53
Zinc finger domain proteins - eg nuclear receptors
Leucine zipper proteins -eg Fos
Helix loop helix -eg Myc

438
Q

What does gene expression in prokaryotes involve?

A

RNA polymerase and sigma factors (starts transcription) for progression from the promoter

439
Q

Prokaryotes: what does the hairpin loop in mRNA do?

A

Stops RNA polymerase processing DNA = termination

440
Q

What does gene expression in prokaryotes involve?

A

Polycistronic (multiple) mRNAs generated from a single gene

Usually proteins / enzymes involved in regulation of a process

Cis regulatory regions and operator regions control initiation of transcription

1 promoter generates multiple genes

441
Q

What are activators of expression?

A

Cis regulatory sequences in promoter and enhancer bind the protein

Trans regulatory factors - ie transcription factors

Control recruitment of accessory factors to RNA polymerase, to control initiation of transcription

442
Q

How does Trytophan repress transcription?

A

It binds to repressor > ACTIVE! This causes a shape change > can bind to operator site > RNA polymerase can’t bind > all genes under expression of promoter are turned OFF
Operon OFF

Absence of trytophan > operon is ON > RNA polymerase can bind! = transcription

443
Q

How do Operons control transcription?

A

Binding of activator to the cis regulatory region recruits RNA polymerase to ‘turn on’ transcription

Operon is ON

Eg: - glucose, + lactose

444
Q

Why are prokaryotic genomes more simpler?

A

Polycistronic mRNAs

445
Q

Prokaryotes have less what?

A

Less non coding DNA and less elaborate control of gene transcription

446
Q

What are common features of eukaryotic and prokaryotic gene control?

A

Consensus sequences
Transcription factors (trans activators)
Ability to inhibit transcription with binding of repressors

447
Q

Eukaryotic transcription summary

A

Non coding DNA in the form of promoters and enhancers controls initiation of transcription

Promoters contain consensus sequences to recruit GTF

The components of the mediator complex control initiation through 3D positioning

448
Q

Why is TFFIID (GTF) essential for transcription initiation by eukaryotic RNA pol 11?

A

Quite long and attaches to multiple parts of promoter

449
Q

RNA Pol 11 cannot work without:

A

Activators
Mediators
Chromatin modifying proteins

450
Q

The eukaryotic enhancer

A

Non coding DNA

Cis regulatory and trans activating factors regulate this area

3D EFFECT that brings a complex of proteins together to the form a mediator complex

451
Q

What are the main readout mechanisms?

A

The recognition of bases and the recognition of DNA shape

452
Q

What does the formation of higher order protein - DNA complexes depend on?

A

Sequence dependant DNA structures that are optimised to promote assembly

453
Q

DNA binding proteins can move to the nucleus after activation.
What are the steps in this process?

A

Movement to the nucleus may require a change in shape or dissociation from another protein

The change reveals the NLS - key to bind to Importins

Then allows the protein to bind to Importins to allow it to move through the NPC and into the nucleus

Once in the nucleus it can dimerise with other proteins and DNA

And therefore can affect gene expression by controlling initiation of transcription

454
Q

How do DNA binding proteins get into the nucleus?

A

Protein synthesis

Ligand binding

Covalent modification - post translational modification

Addition of a 2nd subunit - eg dimerisation

Unmasking - eg of a chaperone protein that may be covering up NLS

Release from membrane - eg protein kinase C that moves into nucleus

455
Q

What are Homeobox (HOX) genes?

A

HOX proteins control body patterning during (foetal) development - eg HOX9 is involved in limb development

They contain homeodomains - contain 3 alpha helices > packed closely together by hydrophobic interactions

456
Q

P53: a b sheet recognition protein

A

Typical tumour suppressor gene - stops us having continuous proliferation

Contains a DNA binding protein made of 2 b sheets - almost every mutation that causes cancer happens in the DNA binding domain!

Forms multimers through its OD domain - can modify its DNA sequence specificity

457
Q

Zinc fingered nuclear hormone receptors

A

Finger like domains that interact with DNA

Regulate processes like bile acid detoxification - eg FXR

Must bind as a dimer (2 of the same protein)

Contents of the dimer are key to sequence specifity and effect

458
Q

What are the functions of p53?

A

Stops the cell cycle but promotes DNA repair

459
Q

Fos - is a leucine zipper that regulates bone

A

Always binds to DNA as dimers

Dimer formation enabled by hydrophobic interactions between alpha helices

Have a globular domain with basic leucine zipper (bZIP) - determines if it has open or closed structure

Activated by phosphorylation - by MAP kinase

460
Q

How is Fos activated?

A

Signalling cascade of kinases > Fos phosphorylation

Heterodimerisation with Jun enables binding to the AP1 response element

Proliferation of fibroblasts and differentiation

461
Q

Myc has a role in cancer (it is often up regulated)

A

Components of the heterodimer control the outcome of the signalling pathways

Myc associated with: cell cycle progression / apoptosis / proliferation / metabolism

Myc can associate with Max. When they bind to M21, they repress genes and promoters!

462
Q

How does dimer formation affect protein concentration?

A

If dimer formed 1st: increase number of sites bound on DNA = quickly increases protein concentration

If dimer takes longer to form: protein concentration increases slowly

463
Q

Bile acids act as …

Retinoic acids act as …

A

Co repressors

Co activators

464
Q

What is precision medicine?

A

An emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment and lifestyle for each person

Ones size fits all approach

465
Q

How is precision medicine different to personalised medicine?

A

Personalised: older term with risk of misleading interpretation
Individual, unique treatment

Precision: treatments for groups of people based on genetic, environmental and lifestyle factors

466
Q

How did we get to precision medicine?

A

Multiple sources of data

Electronic health records

Collaborative data sharing

Extensive research

Education and awareness in multiple levels

467
Q

Why is DNA mostly used in genetic testing and not RNA?

A

Aberrant gene expression or splicing effects in RNA!

468
Q

How can you genetic test for diagnosis? Eg Mendelian disorders

A

Sequencing a single gene - eg cystic fibrosis

Sequencing a panel of genes - eg Kabuki syndrome

The clinical exome

WES and WGS

469
Q

Genetics for screening purposes

A

Not diagnostic!

To identify high risk sub groups who undergo diagnostic test

Statistically quantifying the performance of a test

At different times of life (prenatal, newborn, adulthood)

Must be evaluated: benefits vs costs

470
Q

Genetics of complex traits

Eg type 2 diabetes

A

Complex = multi factorial (polygenic, lifestyle, environment)

Find people at high genetic risk to offer targeted advice

Polygenic risk scores

471
Q

What are polygenic risk scores?

A

Sum the risk alleles of multiple variants, usually weighted by their effects as observed in large scale studies

Population specific!

472
Q

Type 2 diabetes: even with 65 genetic variants the prediction is poor!
Why?

A

Individual effects are modest
Only 10% of genetic predisposition found
Currently over 500 variants across the genome associated with T2D

473
Q

What are pharmacogenetics?

A

How people respond to drugs based on their genetics

Adverse side effects - eg Warfarin

Targeted cancer drugs for specific mutations

Future: the most efficient dose

474
Q

Warfarin is a drug for people at risk for embolism or thrombosis
What were its side effects?

A

Decreases availability of vitamin K which is an essential cofactor in blood clotting

Excessive bleeding in people who have low activity enzymes involved in drug metabolism

475
Q

Gene therapy

A

To treat or cure a disease by modifying a persons genes - replace with a healthy copy, introduce a new gene

Mechanisms - plasmid DNA, viral and bacterial vectors, gene editing technologies

If targeting RNA transcripts > RNA therapeutics

Somatic vs germline

Tightly regulated

476
Q

Examples of gene therapy: Haemophilia B
What is it?

A

A blood clotting disorder causing easy bruising and bleeding
Mutation in the gene causing deficiency of clotting factor IX

477
Q

Ethical issues of gene therapy

A

Prenatal screening

Incidental findings - screen only selected genes and not the whole genome

Baby editing

Germline gene therapies

The safety of gene therapies

Costly treatments

Who gets treatment

478
Q

What are SNPs?

A

A single nucleotide substitution characterised by its alleles and position in the chromosome

Most SNPs (99%) are bi allelic (two alleles) and uncommon (MAF <5%)

479
Q

Indels are the … type of variant and typically exist in …

A

Second most common

Two alleles

480
Q

The vast majority of SV are …

A

Insertions, deletions and duplications

481
Q

SV are responsible for … of all protein truncating events in the genome

A

25-30%

482
Q

Up to 4% of individuals carry …

A

Mega base pair SVs

483
Q

Copy number variants are the … type of SV in the population

A

Most frequent

484
Q

There are over … in the human genome, of which at least 373 000 are VNTR

A

55 million tandem repeats.

485
Q

Short VNTR called … occur in multiple alleles and have a high mutation rate

They are … in the human genome

A

Micro satellites
Abundant

486
Q

VNTR contribute to … in the expression levels of 12,494 genes through multiple mechanisms

A

Variation

487
Q

Over 40 Mendelian disorders , such as Huntington disease, are attributed to …

Most are caused by … of tri nucleotide coding repeats

A

STR mutations
Large expansions

488
Q

Haplogroup analysis of … and … can trace the origin of Homo sapiens and their expansion out of Africa

A

Mitochondria
Y chromosome DNA

489
Q

… can help estimate when the first humans emerged from the last common ancestor with apes

A

Molecular clocks of DNA

490
Q

What have humans developed through out migration?

A

Adaptations to new environments

491
Q

What is the human genome?

A

The genome is the set of all genetic information of an organism

492
Q

Do you think all genetic info is stored in DNA molecules?

Interspecies somatic cell nuclear transfer example

A

DNA is the only fundamental molecule that stores genetic info!

Good evidence that the chromosome and DNA are good candidates for being molecules that start genetic information

Also evidence that mitochondria has DNA, but NOT fundamental to define your species!

493
Q

Trans generational epigenetic inheritance: mouse example.

What happened to the mice offspring?

A

Mother mouse was induced with stress> didn’t properly groom offspring

So 1st generation didn’t properly groom their offspring

2nd and 3rd generation still didn’t groom offspring

So besides DNA, mice will also inherit behaviour from their mother!

494
Q

What was the effect of the mother mouse being stressed during pregnancy?

A

Will have the phenotype of NOT grooming children properly > problem transferred to next generations!

DNA is still the same > but what changed is the way the DNA spreads!

So genome is still the DNA, but the way we read the genome can change depending on circumstances

495
Q

Mitochondria is always inherited from …

Y chromosome inherited from …

A

Mother
Father

496
Q

Why are introns useful?

(26% of genome composition)

A

Useful as allow recombination to occur

497
Q

What are transposons?

A

Mobile DNA elements that can insert themselves into another position of DNA sequence

498
Q

What is the role of transposons in bacteria?

A

Are responsible for most antibiotic resistance in bacteria

Can jump from chromosomal DNA to plasmids and vice versa
This facilitates the horizontal transfer of genetic material between species - including antimicrobial resistance genes

499
Q

What characteristics enable transposons to jump between species?

A

Transposons include enzymes required for their movement in the coding sequence

They jump to any DNA molecule

Transposed plasmids and viruses may conjugate / infect another organism

New transposons can integrate into the human genome

500
Q

Why is so much of our DNA transposons?

A

Because it keeps copying and replicating our genomes!

501
Q

What prevents transposons from colonising the entire genome?

A

The human genome is relatively stable because most of the transposons are silent!

502
Q

Why do defective transposons contribute to junk DNA?

A

Transposons are also called ‘junk DNA’ because they replicate without an apparent benefit to the host!

503
Q

What traits evolved from transposons?

A

The adaptive immune system

The placenta

504
Q

How do transposons contribute to genetic variation?

A

Transposition may re arrange DNA

Transposons facilitate horizontal gene transfer between species

Deactivated transposons accumulate mutations over generations

505
Q

Mutation - processes that increase variation

Examples?

A

DNA replication and repair errors

Horizontal gene transfer

Transposition

506
Q

Selection - processes that decrease variation

Examples?

A

Natural selection

Genetic drift

507
Q

What is an Indel?

A

An insertion or deletion of up to 50 nucleotides in a specific location of the DNA sequence

508
Q

Cystic fibrosis is a clinical example of an indel
What is it caused by?

A

The 3 nucleotides CTT are deleted in people that develop Cystic Fibrosis

Therefore Phenylalanine is NOT in the amino acid sequence > conformation change in protein > exchange of ions in surface of bronchi is affected > people develop disease later on in adulthood!

509
Q

What are structural variants? SV

A

Are large >50bp rearrangements of DNA segments

510
Q

What is the median number of SV that a human has?

A

7,439

511
Q

What characteristic distinguishes an indel from an insertion or deletion?

A

The length, indels are <50 bp

512
Q

Why does the Y chromosome become degenerated?

Why does satellite DNA easily accumulate there?

A

Because recombination doesn’t occur there! (Heterochromatin)

A lot of mutations occur in Y chromosome that don’t occur in other chromosomes > mostly junk!

513
Q

Autosomal DNA (Chr1 - 22) is inherited from …

A

Both parents

514
Q

Mitochondrial and Y chromosome DNA is inherited from …

A

1 parent only

515
Q

What is a halo type?

What does halo type analysis reveal?

A

A group of alleles that are inherited together from the same parental chromosome

Reveals relatedness between individuals

516
Q

How have Homo sapiens adapted to high altitudes?

A

Environmental stresses: lower atmospheric pressure, lower oxygen, lower temps and changes in diet

Evolutionary responses: positive selection of variants conferring adaptation to environment

517
Q

What may Mendelian diseases be caused by?

Example?

A

Coding and non coding SNPs may have phenotypic effects - ie may cause Mendelian diseases

Eg - Phenylketonuria PKU is an autosomal recessive disease caused by substitution in the sequence of the phenylalanine hydroxylase gene

518
Q

The first draft of the complete sequence of the human genome…

A

Included DNA from multiple volunteers

Published in 2001

Human genome project

519
Q

What do most phenotypes display?

A

NON - Mendelian patterns of inheritance

520
Q

Apart from complex traits, how can most inheritance patterns be studied?

A

Using Punnet tables

521
Q

How can complex traits be analysed?

A

With quantitative genetics methods, like heritability

522
Q

What can twin studies estimate?

Using what?

A

Phenotypic heritability, without genotyping individuals

Using Falconers Formula

523
Q

What are Mendelian traits?

Eg pea colour

A

Traits explained by mutations in a single gene

Do NOT have intermediate phenotypes - are either dominant or recessive!

Phenotype is NOT influenced by environment or epigenetics

524
Q

What are Co dominant traits?

Eg ABO blood group

A

Occurs when a phenotype is controlled by a gene with multiple alleles

2 or more alleles are expressed simultaneously

525
Q

What is a polygenic trait?

Eg eye colour

A

Causes by multiple genes

Phenotype can show a range of continuous variation

The extremes tend to be less frequent - induces almost a normal distribution

526
Q

What is Epistasis?

Eg mouse / dog coat colour

A

Genetic phenomenon when the effect of a mutation in a gene is dependant on mutations on other genes!

527
Q

What is pleiotropy?

A

A phenomenon where mutations on a gene affect 2 or more apparently unrelated phenotypes!

Eg a single mutation on the B globin gene may cause sickle cell disease - which is protective against malaria.
Therefore, the SAME gene is a risk factor for sickle cell anaemia and a protective factor for malaria!

528
Q

What are complex traits caused by?

A

Genetics and a multiplicity of unknown environmental effects

Cannot be described by a Punnet square

529
Q

What is heritability? (H2)

A

The proportion of variance in a phenotype explained by genetic factors

Does not quantify whether a trait is genetic or not

530
Q

What may single nucleotide substitutions on the coding sequence influence?

A

RNA and amino acid sequence

531
Q

What may non exonic SNPs and Indels in the gene body do?

A

Insert stop codons and disrupt the protein!

532
Q

Several diseases are caused by … via multiple …

A

SVs
Mechanisms

533
Q

Diseases caused by copy number variants:

A

Velo cardio facial syndromes

Williams Beuren syndrome

Prader Willi and Angelman syndrome

534
Q

Missesense =

A

Single amino acid substitution

535
Q

Nonsense =

A

Results in a shorter unfinished protein product typically by substitution of an amino acid with a STOP codon

536
Q

Frameshift =

A

Results in alteration of all amino acids after an insertion or deletion

537
Q

Coding and non coding SNPs may cause monogenic and complex disease
An example?

A

Phenylketonuria - PKU - monogenic disease

Caused by substitutions in the sequence of PAH gene that codes for phenylalanine hydroxylase enzyme

Coding SNPs in exons and non coding SNPs in the introns, 5’ and 3’ UTR of phenylalanine hydroxylase gene causes PKU

538
Q

GWAS = genome wide association studies

General characteristics?

A

Similar to linkage studies but in unrelated individuals

Better than linkage studies at detecting weak genetic effects

To identify personal genetic disease risk

To identify pharmacogenetic variants (drug response)

Identity variants contributing to gene environmental interactions

539
Q

What has enabled GWAS?

A

Advances in genotyping technologies and lowered costs

Advances in software

Biobanks

Large scale projects - HGP, international HapMap project

540
Q

What is the focus on in GWAS?

A

Single nucleotide polymorphisms SNPs

541
Q

Key concepts in GWAS?

A

SNP

Minor allele frequency MAF

Hardy Weinberg equilibrium

Linkage disequilibrium

Haplotypes

542
Q

What is a single nucleotide polymorphism?

A

A DNA sequence variation occurring when a single nucleotide (A, T, C or G) in the genome differs between members of a species OR between paired chromosomes in an individual at a particular locus

543
Q

What is the minor allele frequency?

A

The frequency of the SNPs less frequent allele in a given population

Population specific

544
Q

What is the hardy Weinberg equilibrium?

A

Both allele and genotype frequencies remain constant in a population unless specific disturbing influences are introduced
(mutation, migration, non random mating)

545
Q

Linkage disequilibrium and haplotypes

A

The non random association of alleles at 2 or more loci so they are inherited together more frequently than expected by chance

Genetic correlation between the markers

Observed in various regions of the genome

Decreases with physical distance

Segments of high LD = haplotype blocks

LD structure is population specific

546
Q

The TopMed project has identified over 300M SNPs in humans

How could you cover all common variants?

A

Using tagSNP selection

547
Q

Quality Control QC

A

The most important and time consuming part of GWAS!

To reduce systematic bias

548
Q

What are QQ plots?

A

Used as a diagnostic tool by plotting observed p values against expected

If p values are close to the grey line (null hypothesis) this implies there a few systematic sources of spurious association

Deviation of small p values from null line indicates possible associations

Log scale helps to emphasise the smallest p values

549
Q

What are possibilities for an identified association in GWAS results?

A
  1. There is a causal relationship between the SNP and trait
  2. The marker is in linkage disequilibrium with a causal locus
  3. False positive
550
Q

There are many potential sources of systematic error in GWAS that might lead to false positive results

How could you overcome this?

A

Genotyping quality control is particularly important

551
Q

How could you find the missing heritability in GWAS?

A

Increase sample size - biobanks

Gene x environment interaction studies

Analysis of rare variants

Analysis strategies increasing power - eg multi phenotype analysis

552
Q

Post GWAS

A

Identify the causal variant to understand molecular mechanisms and pathways behind them

Most identified SNPs are in NON coding regions, could still have consequences for nearby genes - enhancer elements,
DNase hypersensitive regions

553
Q

What is DNA sequencing?

A

Working out the order of the 4 bases (A, T, C, G) in fragments of DNA

Usually by amplified PCR or DNA cloning

Increasingly sophisticated and affordable

554
Q

What is RNA sequencing?

A

RNA is sequenced indirectly through the sequencing of DNA

555
Q

What is protein sequencing?

A

Time consuming and requires relatively high amounts of protein

Increasingly replaced by modern methods

556
Q

DNA sequencing includes single read sequencing and massive parallel sequencing.
Examples of both?

A

SR:
Maxam Gilbert method - based on chemical degradation. Now obsolete.

Sanger method - based on primer extension chain termination. Still used but dominating method is NGS

MPS:
Next generation sequencing NGS - based on primer extension, fully automated, a revolution in progress

557
Q

Sanger sequencing

A

DNA sequencing with chain terminating inhibitors = dideoxy sequencing

Used for sequencing SINGLE genes and fragments of DNA - mutation screening in specific genes, validations of findings from NGS

Highly accurate

558
Q

What are the steps of pre sequencing?w

A
  1. Need to produce multiple copies of DNA - clone DNA fragment into plasmid and grow in E.coli OR amplify DNA fragment by PCR
  2. Denature the sequence by heating (or adding NaOH) to produce single stranded DNA
  3. Prepare DNA polymerase, primer, dNTPs and ddNPTs
559
Q

Primer extension

A

DNA polymerase will start synthesising a complementary stand

Starting from the primer (adding bases)

Forms a complementary copy to the template strand - extending the primer therefore

560
Q

What are dideoxyribonucleic triphosphates ddNTPs?

A

Terminator nucleotides

Modified version of the normal DNA building blocks (dNTPs)

Use the SAME bases (ATCG) but the sugar is modified!

Wherever a ddNTP has been incorporated, DNA synthesis CANNOT proceed any further

561
Q

How does ddNTP terminate DNA synthesis?

A

Lack of the 3’ OH group in the deoxynucleotide prevents the formation of the phosphodiester bond

562
Q

Termination of DNA synthesis process

A

There are multiple strands due to DNA amplification

There is an excess of normal dNTPs against the amount of ddNTPs (100:1)

These compete against eachother in the DNA synthesis

Termination happens at different places at different strands

The result is a set of DNA sequences varying in length - each ending with a ddNTP

563
Q

Sanger sequencing

A

Primer, DNA polymerase and a mix of normal (unlabelled) dNTPs and labelled ddNTPs

Label used to be radioactive BUT today fluorescent dyes are used

The correct order is reached through gel electrophoresis and fluorescence detection

In genotyping where only a SINGLE SNP is genotypes, process is similar but NO normal unlabelled dNTPs are added!

564
Q

How are nucleic acids separated by size in slab gel electrophoresis?

A

Nucleic acids carry numerous negatively charged phosphate groups

They will migrate towards the positive electrode when placed in electric field

Porous gel acts as a sieve - small molecules pass through easily than larger fragments

564
Q

How are nucleic acids separated by size in slab gel electrophoresis?

A

Nucleic acids carry numerous negatively charged phosphate groups

They will migrate towards the positive electrode when placed in electric field

Porous gel acts as a sieve - small molecules pass through easily than larger fragments

565
Q

How did Sanger sequencing work in 1977-1985?

A

Radioactive labelling requiring 4 separate reaction tubes for each ddNTP

Separated individually on large slab electrophoresis

X ray film and dark room manipulation

Manual reading and feeding into computer

1.5 days from setup to results

565
Q

How did Sanger sequencing work in 1977-1985?

A

Radioactive labelling requiring 4 separate reaction tubes for each ddNTP

Separated individually on large slab electrophoresis

X ray film and dark room manipulation

Manual reading and feeding into computer

1.5 days from setup to results

566
Q

How did Sanger sequencing advance from 1986 onwards?

A

Fluorescent dye for labelling, use of a mixed reaction tube containing ALL 4 ddNTPs

Automated optical detection system using a laser

Direct automated entry of DNA base sequence into computer

Still time consuming due to handling of gel plates

567
Q

How is capillary gel electrophoresis more advanced compared to slab gel electrophoresis?

A

Slab gel is manual - slow and prone to human error

Capillary is largely automated

Gel is not run for a finite time, fluorescent labelled DNA samples migrate through gel

Allows longer reads up to 1000 bases - each fragment is allowed to run to bottom of gel where resolution is highest

Faster and cheaper

568
Q

Pros and cons of Sanger sequencing

A

Highly accurate sequences - about 99.95%

Several hundred bases long 800-1000bp

Gel electrophoresis is NOT suitable for handling large numbers of samples at a time because it is not fully automated

Therefore not suitable to genome sequencing

569
Q

Next generation sequencing NGS

A

Also known as massively parallel sequencing - meaning it sequences millions of DNA fragments at same time

From 2005 onwards - a technological revolution

Vast increase in amount of sequence per data run (the sequence throughput) = dramatic loss in cost

Moving from sequencing single genes and exons to: whole genome and exome sequencing, methyl seq, CHIP seq and ribo seq

570
Q

What is throughput?

A

The amount of sequence data processed in one run

571
Q

What is the read length?

A

Length of DNA fragments measured in nucleotides

Short read lengths = high throughput

572
Q

What is read depth?

A

The sequence coverage - how many times each sequence is represented

Important for small read lengths for genome assembly

572
Q

What is read depth?

A

The sequence coverage - how many times each sequence is represented

Important for small read lengths for genome assembly

572
Q

What is read depth?

A

The sequence coverage - how many times each sequence is represented

Important for small read lengths for genome assembly

573
Q

What is genome assembly?-

A

Aligning and merging the sequenced pieces to make sense of the sequenced genome

574
Q

What is second generation sequencing ?

(Based on amplified DNA templates)

A

From short (35 nucleotides) to medium length sequences (up to 800 nucleotides)

High to very high sequence throughput

Quite a high rate of sequencing errors in individual reads

575
Q

What are the commonly used second generation sequencing platforms?

A

Roche / 454 pyrosequencing

The ABI SOLID technique

Illumina / Solexa sequencing

Ion torrent systems

576
Q

Sequencing with emulsion PCR

A

Uses bead surfaces, water and oil

Allows simultaneous amplification of each sequence without risk of contamination

Each beads acts as a microreactor for PCR, each containing one strand of DNA

The terminators are reversible - after chemical de protection synthesis can continue!

577
Q

Third generation sequencing (single molecule sequencing SMS)

(Based on unamplified DNA)

A

Long sequences (thousands of nucleotides) - important for assembly of genomes from newly sequenced species, and for distinguishing large scale variations

Release of protons which is recorded as an electric current

Avoids problems related to DNA amplification - eg under or over representation of DNA

Simple and cheap technology - small portable machines eg Oxford Nanopore

High error rates

578
Q

Single cell sequencing

A

Works on cell populations > resulting data are aggregate values

For understanding cell to cell variation and identifying new cell types

A catalog of human cells - stable cell properties / cell positions / lineage relationships

Widely employed in cancer research

579
Q

Analysis of NGS data

A

Output files contain millions of short (100bp) reads (2nd generation) or longer reads (3rd generation)

Reads are mapped to reference genome if species is known

Variants are identified

Requires several software tools and computational skills

580
Q

Sanger vs NGS

A

Sanger:
Cheap, fast, simple
Highly accurate
Gives an answer about a single, specific question

NGS:
Getting cheaper with more technologies
More error prone
Highly versatile - WGS, WES, RNA seq, methyl seq etc
Computationally laborious

581
Q

What is a property of DNA polymerase that is important for its proofreading function?

A

3’ to 5’ exonuclease activity

582
Q

The role of nucleotide excision repair is to correct error in the DNA introduced by…

A

Exposure to UV light

583
Q

What is CORRECT regarding tumour suppressor gene?

A

Loss of function mutations can inactivate them

Loss of function mutations can contribute to cancer

584
Q

What is NOT true for cancer cells?

A

Cancer cells show growers repression by growth suppressive signals

585
Q

Viruses can infect cells to cause oncogenic transformation.
What is NOT true regarding this process?

A

Viral borne cancers can be transmissible

586
Q

Mutations in TCA cycle enzyme: isocitrate dehydrogenase can change its activity
What is the name of the product formed as a result of this reaction?

A

2 hydroxy glutarate

587
Q

What element covers the greatest part of the human genome?

A

Repetitive and transposable elements

588
Q

Is genetic info found in all organisms?

A

YES

589
Q

What cells contain the genetic material?

A

Zygotes
Gametes
Brain

590
Q

How many SNPs have been identified in the human genome?

A

100 million

591
Q

What is the maximum dosage of a bi allelic SNP?

A

2

592
Q

What is a large structural variant?

A

Deletion

593
Q

What is a variable number tandem repeat?

A

A short DNA sequence repeated in tandem that vary in number

594
Q

A polygenic trait is controlled by …

A

Multiple variants / multiple mutations

595
Q

Is it possible to carry genes for a disease you do not have?

A

Yes!

Eg cystic fibrosis in Europeans - recessive allele carried by 0.8% of population

596
Q

Is the offspring of bodybuilders naturally more muscular?

A

No!

Genetic info has not changed

597
Q

What is heritability?

A

The proportion of phenotype variance explained by genetic factors

598
Q

Does heritability indicate the proportion of the trait that is due to genetics?

A

No!

599
Q

What protein bridges the 5’ and 3’ of the mRNA through protein protein interactions?

A

eIF4G

600
Q

What statement regarding eIF2 is FALSE?

A

eIF2 is a guanine exchange factor for eIF2B

601
Q

What is true about eIF4E?

A

The mTOR signalling pathway can regulate eIF4E activity

602
Q

What is FALSE regarding the m6A RNA methylation?

A

m6A and m1A modifications are mutually exclusive on an mRNA

603
Q

What is FALSE about eukaryotic ribosome subunits?

A

Contain the same number of rRNAs than those of prokaryotic ribosomes

604
Q

What is an mRNA quality control pathway?

A

Non stop decay

605
Q

What is FALSE regarding mRNA surveillance pathways?

A

UAG is always a premature termination codon

606
Q

Vanishing white matter disease …

A

Is a result of diminished ability to recycle eIF2-GDP to eIF2-GTP

607
Q

Dosage compensation in flies is achieved by a translational control mechanism mediated by…

A

Translation repression by SXL

608
Q

What is FALSE about HRI?

A

HRI is an ER resident eIF2 kinase that responds to unfolded proteins

609
Q

Sanger sequencing is used for SINGLE genes

A

Sequencing process: ddNTPs

Gel electrophoresis

Highly accurate

609
Q

Next generation sequencing for MULTIPLE genes / gene regions / genome

A

Second vs third generation

Genome assembly

WGS vs WES vs RNAseq

Analysis of NGS data

610
Q

GWAS

A

A hypothesis free approach that has revolutionised complex traits genetics

Testing millions of imputed SNPs for association between the genome and the trait of interest

Results are combined from multiple studies (meta analysis) to provide evidence

Quality control PRIOR to analysis is very important to avoid systematic errors coming from data!

The difficult starts after GWAS to understand the underlying biology

Although successful, a lot of ‘missing heritability’ remains

610
Q

Precision medicine

A

Genetic testing for Mendelian disorders

Genetic testing for screening

Polygenic risk scores PRS for complex traits to help prediction

Genetics to help understand response to drugs (pharmacogenetics)

Gene therapy

Challenges and future perspectives

611
Q

Polygenic risk score graphs

A

Normal distribution

Population specific

Important to go genetic studies in different ancestries as genetics may differ in different ancestries

611
Q

Polygenic risk score graphs

A

Normal distribution

Population specific

Important to go genetic studies in different ancestries as genetics may differ in different ancestries

611
Q

Polygenic risk score graphs

A

Normal distribution

Population specific

Important to go genetic studies in different ancestries as genetics may differ in different ancestries

612
Q

ROC curve

A

For prediction purposes

Try to maximise area under the curve > the higher it is, the better the test!

Optimal combination of sensitivity and specificity

613
Q

Factors affecting PCR

A

Melting temp Tm - temp that dictates the annealing step

GC content - Lower the number of Gs and Cs in the primer, the lower the melting temp!

Salt content - sodium acetate

Other buffer components - like denaturant > may change structure of DNA, may not allow base pairing!

614
Q

Quantitative PCR

A

Tells you how much mRNA was there to start with and can compare it to other mRNAs in sample

614
Q

Quantitative PCR

A

Tells you how much mRNA was there to start with and can compare it to other mRNAs in sample

615
Q

Electrophoretic mobility shift assay EMSA

A

Detects proteins binding to specific sequences (response elements)

Always a band at the bottom because that’s where the probe is and the probe hasn’t bound !

You will never get every piece of DNA bound by protein > some left over

616
Q

What are ATP dependant processes in epi genetics ?

A

Nucleosome eviction

Nucleosome sliding

617
Q

Histone chaperones and ATP dependant DNA remodellers are required for what process?

A

Nucleosome sliding

618
Q

What is the function of Histone tail modifications?

A

To allow transcription factors to bind to a promoter and therefore induce gene expression

To bring arts of DNA closer together in a complex that might enable gene expression to occur

To change interaction of Histones with DNA therefore changing chromatin structure

To work in concert with DNA methylation to manipulate the access of DNA to transcription factors and transcription machinery

619
Q

What methods enable a scientist to identify areas of DNA that might regulate gene expression?

A

Hi-C and ChiA-PET

620
Q

In a ChiP assay, what readout method enables identification of transcription factors part of a complex?

A

Western Blot

621
Q

What enzymes carry out de novo DNA methylation which occurs mainly in embryonic development?

A

DNA methyl transferase 3 (DNMT3a and b)

622
Q

What modifications can occur at a lysine in Histone tails?

A

Ubiquitinylation

Di methylation

Acetylation

Tri methylation

623
Q

What is the name of the mechanism where nucleosomes are removed to transform DNA from heterochromatin to euchromatin structure?

A

Nucleosome eviction

624
Q

Deamination of DNA bases can change…

A

Cytosine to uracil

625
Q

Cancer cells often display the Warburg effect. It means:

A

They prefer to carry out aerobic glycolysis even in the presence of oxygen!

626
Q

What is INCORRECT about proto oncogenes?

A

Proto oncogenes are derived from tumour suppressor genes

627
Q

The 2019 Nobel prize in medicine and physiology was awarded for…

A

How cells sense and adapt to oxygen availability

How kidneys stimulate erythrocyte production by erythropoietin up regulation

How hypoxia inducible factor is stimulated upon low oxygen

How cancer cells form new blood vessels to disseminate themselves

628
Q

What defines a cancer hallmark

A

An exclusive characteristic feature of a cancer cell

629
Q

Retinoblastoma is a … that functions by acting as a … regulator of transcription factor …

A

Tumour suppressor

Negative

E2F

630
Q

What is correct about the Her2 gene?

A

A valine to glutamine mutation in this gene causes constitutive activation

631
Q

What is the product formed as a result of the mutation in the TCA cycle enzyme isocitrate dehydrogenase?

A

2 keto glutarate

632
Q

UV light directly damages DNA by…

A

Causing thymine thymine dimers

633
Q

DNA glycosylase regulates the initial step of…

A

Base excision repair

634
Q

What is true about Benz(a)pyrene?

A

It undergoes P-450 mediated activation in lungs causing G to T conversion

635
Q

What is incorrect in regard to viruses and oncogenic transformation?

A

Viruses can mutate cellular proto oncogenes into oncogenes

636
Q

As a sensor of DNA damage, ATM is a … which can … p53 through …

A

Kinase

Activate

Phosphorylation

637
Q

What is incorrect about cancer cells?

A

Cancer cells show growth repression by growth repressive signals

638
Q

What defines the DNA polymerase proof reading activity?

A

It also has a 3’ to 5’ exonuclease activity to remove any incorrect base pairing

639
Q

What signal can activate p53 function?

A

Lack of nucleotides

640
Q

What is correct regarding tumour suppressor gene?

A

Loss of function mutations can inactivate them!

641
Q

What is incorrect regarding aerobic glycolysis?

A

Aerobic glycolysis produces HIGH ATP output

642
Q

What is true regarding the androgen receptor?

A

It’s a nuclear receptor that drives prostate cancer

Found inactive in cytoplasm and activated by androgen binding

Regulates gene expression in prostate epithelial cells

A target of anti androgen is drugs in the clinic

643
Q

What is correct about p53?

A

It’s a tumour suppressor gene that opposes uncontrolled cell growth caused by oncogenes

644
Q

HIF1a is … under … oxygen conditions by … mediated …

A

Stabilised

Low

Prolyl hydroxylase

Covalent modification

645
Q

What is NOT a characteristic of cancer cells?

A

Limited proliferation

646
Q

What is correct about 2 hydroxyglutarate. 2HG?

A

It is a metabolite specific to cancer cells!

Oncometabolite

647
Q

Loss of function mutations in tumour suppressor genes can result in…

A

Tumour formation

Inability of a tumour suppressor to inhibit proliferation of a cell with DNA damage

Uncontrolled growth of cells that harbour oncogenes

Loss of balance between cell proliferation and cell death

YEAR 2 - Genetics (1 decks)

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