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GMC > Block 1 - genetics > Flashcards

Block 1 - genetics Flashcards

1
Q

what factors regulate gene expression

A

TFs
chromatin and histone modification
nuclear organisation

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2
Q

what do ES differentiate into in the presence of low retinoic acid

A

cardiomyocytes

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3
Q

what do ES differentiate into in the presence of high retinoic acid `

A

neuronal cells

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4
Q

what are the marker genes for ES cells, cardiomyocytes and neurons and what are their functions

A

ES - OCT4 - TF for self renewal
cardiomyocytes - cardiac troponin C (TNNC) - binds calcium ions to activate muscle contraction
neurons - MAP2 - stabilises microtubules in the dendrite

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5
Q

what is quantitative/real time PCR used for

A

it is used to amplify and quantify the amount of a specific RNA in a sample

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6
Q

what are the steps in RT qPCR

A
  1. culture cells and treat them (e.g. retinoic acid)
  2. purify RNA from cells
  3. use RT to synthesise cDNA from RNA (RAN can’t be used in PCR). mRNA has poly A tail so we use oligo (dT) or random hexamer primers. Primers anneal and RT fills in cDNA which is then separated from RNA using alkali.
  4. PCR using cDNA
  5. fluorescent dye binds to DAN and fluoresces - fluorescence is measured and doubles with every PCR cycle
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7
Q

what material are required for RT qPCR

A 
RT
primers 
gene and reference gene 
dNTPs 
buffer 
high quality RNA 
PCR mix 
dye - SYBR
fluorescence measuring machinery and computer for analysis 
Taq pol
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8
Q

what is semi-quantitative PCR

A

after a set number of PCR cycles an agarose gel electrophoresis is carried out and DNA is detected by staining

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9
Q

how does the fluorescence work in RT q-PCR

A

SYBR green fluorescent dye intercalates into dsDNA

geometric phase data is collected for accurate quantification of starting material

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10
Q

what is the Ct value

A

PCR cycle number where the fluorescence becomes greater than the threshold

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11
Q

how do we normalise quantification calculations

A

they are normalised to a reference gene (don’t change in the experiment) which is used to control for any experimental variability e.g. sample preparation, RNA isolation, RT efficiency, PCR set up and efficiency

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12
Q

give 3 examples of reference genes

A

B-actin
gadph
BIII-tubulin

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13
Q

describe the 3 main stages in transcription

A
  1. initiation - polymerase binds to the promoter, forming a TF bubble near the start site. RAN polymerase catalyses phosphodiester linkage of 2 initial rNTPs
  2. elongation - RNA pol advances 3’ –> 5’, melting the DNA duplex and adding rNTPs to the forming RNA (forming in 5’ –>3’ direction)
  3. termination - at the stop site the RAN pol releases completed RNA and dissociates form DNA
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14
Q

what are the 3 types of RNA, what is their function, what are they made by and what is their proportion

A

rRNA - made by RNA pol I - ensures translation accuracy - 80%
mRNA - made by RNA pol II - translated into peptide - 5%
tRNA - made by RNA pol III - brings amino acids to the ribosome - 15%

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15
Q

what is RNA pol II transcription initiation regulated by

A

TFs that bind to the promoter region in DNA and recruit coactivators and RNA pol II

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16
Q

name and describe 3 gene regulatory elements

A

promoter proximal elements - activators/repressors that bind to the promoter
enhancer elements - loop over and interact with the factors bound at the promoter
TATA box - DNA sequence that recruits basal/general TFs found at most pol II genes, they recruit and activate

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17
Q

TFs are modular and often act as ………..

They have ….. domains called …….

A

dimers
2
activation domain and DNA binding domain

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18
Q

what is the serum response factor

A

it is a TF that binds to the serum response element in the promoter
the core binding sites for the SRF are 4-12bp and often palindromic due to the TFs often being dimeric

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19
Q

what morphological change can happen to DNA when TFs bind

A

it can bend

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20
Q

how are TFs recruited

A

the TATA box recruits TFIID which recruits other basal or general TFs

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21
Q

what does TFIID consist of

A

TATA binding protein (TBP) and TBP associated factors (TAF)

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22
Q

what is the role of the pre-initiation complex

A

it positions RNA pol at the transcription start site and allows for phosphorylation of the polymerase c-terminal domain

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23
Q

what is the pre-initiation complex made of

A

TFs
mediator complex (coactivator)
mRNA

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24
Q

how is the pre-initiation complex formed

A
  1. sequence specific and basal TFs recruit coactivators e.g. the mediator complex which is required for nearly all RNA pol II transcribed genes
  2. the mediator complex acts as a bridge between TFs, basal TFs and the c-terminal domain of RNA pol II
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25
Q

describe 3 ways that TF activity can be regulated

A
  • conformation alteration through ligand binding/phosphorylation (alter ability to bind DNA, interact with proteins, nuclear localisation)
  • expression level alteration
  • alter DNA binding site accessibility
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26
Q

how is the ELK1 TF activated

A

phosphorylation

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27
Q

how is the CREB TF activated

A

ligand activation

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28
Q

what are MAPK pathways and what do they drive

A

mitogen activated protein kinase pathways

- they drive proliferation or stress responses

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29
Q

what are the 3 basic steps of an MAPK pathway

A
  1. growth factors bind to cell surface receptors
  2. receptor activation –> phosphorylation cascade –> amplification of signal
  3. at the bottom of the cascade TFs get phosphorylated that drive transcription
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30
Q

what happens when MAPK are constitutively activated and where might this be seen

A

transcription is constitutively active so growth is continuous. this is often seen in oncogenic cancer mutations

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31
Q

what is mitogen

A

growth factor

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32
Q

EGF signalling leads to ……. phosphorylation and …. ……..…. ultimately leading to the formation of the PIC and transcription

A

ELK1

DNA binding

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33
Q

how do we produce antibody

A

we inject an adjuvant bound antigen into host animal and harvest antibodies from blood after immune response has been generated

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34
Q

what is SDS

A

an anionic detergent used to denature protein samples and coat them in a negative charge

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35
Q

on what basis are proteins separated in sds page

A

based on size

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36
Q

the sds page gel is vertical/horizontal

A

vertical

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37
Q

in a Coomassie stain of an sds gel highly expressed proteins have thicker/narrower bands

A

thicker

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38
Q

describe the process of western blotting

A
  1. transfer proteins from sds page gel onto membrane using electric current (blotting)
  2. incubate membrane with Ab specific for protein being studied
  3. secondary Ab bound to HRP enzyme is added which recognises the primary Ab and amplifies the signal
  4. HRP substrate added and light emitted is detected by CCD camera
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39
Q

what do western blots show

A

size of the band recognised
Ab specificity
relative amounts of proteins in samples
quality of protein in sample

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40
Q

what is GADPH

A

a loading control

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41
Q

what is immunohistochemistry and how quantitative is it

A

it provides spatial information and uses Abs to detect proteins in cultured cells or tissues often visualised using secondary Abs with fluorescent tags
it is semi quantitative

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42
Q

describe the steps in DNA folding

A 
double helix
beads on a string (nucleosomes)
chromatin fibre of packed nucleosomes 
topologically associated domains 
interphase chromosome 
metaphase chromosome
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43
Q

what is a nucleosome

A

DAN wrapped around 8 histone proteins
there are 4 histones and there are 2 of each in each nucleosome
146bp of DNA is wrapped around the octamer twice

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44
Q

what are histone tails

A

they contact linker DNA and other nucleosomes and are important in chromatin folding and cofactor recruitment

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45
Q

in what 2 ways does chromatin regulate gene expression

A

folding controlled - (access to DNA)

platform for a range of post translational modifications - (accessibility and TF/RNApol recruitment)

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46
Q

what are the 3 core histone tail post translational modifications

A

acetylation
phosphorylation
methylation

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47
Q

why is histone tail modification of significance

A

the modifications bind cofactors which cause activation/folding/repression

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48
Q

describe histone tail acetylation

A

promotes transcription
occurs on lysine residues by HAT (using acetyl coA) which are recruited by TFs. we see hyperacetylation of histone n-terminal tails

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49
Q

describe histone tail deacetylation

A

inhibits transcription

occurs on lysine residues by HDAC which are recruited by repressive TFs and we see deacetylation in the TF vicinity

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50
Q

what are acetylated residues bound by (what is the reader)

A

bromodomain containing proteins (high affinity where multiple acetylated sites exist in proximity). coactivators with bromodomains promote binding of other TFs and the mediator complex leading to RNA pol II recruitment and pic formation

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51
Q

what is constitutive heterochromatin

A

chromatin that is always highly condensed into heterochromatin

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52
Q

where is constitutive heterochromatin found

A

centromeres and telomeres

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53
Q

what is DNA methylation

A

works with repressive histone modifications to condense and silence chromatin

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54
Q

describe histone tail methylation

A

lysine residues can be mono, di or tri methylated by HMT (uses s-adenosyl methionine) which can promote or suppress gene expression depending on the location

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55
Q

describe histone tail demethylation

A

KDM removes methyl groups from lysine residues

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56
Q

what reader binds methylated lysine residues

A

chromodomain containing proteins - can be associated with activation or repression

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57
Q

describe euchromatin

A

less condensed, active genes, gene promoters have active histone modifications, gene promoters are not methylated, replication occurs throughout the S phase, acetylated histones

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58
Q

describe constitutive heterochromatin

A

highly condensed, repressive histone modifications, methylated DNA, no meiotic recombination, replicated in late S phase, non-coding RNA important for centromeric chromatin

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59
Q

describe facultative heterochromatin

A

condensed, inactive genes, repressive histone modifications, methylated DNA (where silenced), replicated in later S phase, non-coding RNA may be involved in repressive regions

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60
Q

if genes are active is the chromatin in the form of euchromatin or heterochromatin

A

euchromatin

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61
Q

where are rRNA genes transcribed

A

nucleolus

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62
Q

what are lamin associated domains

A

heterochromatic regions of chromosomes associated with lamins of the nuclear membrane. most of the genes are silenced. genes move in and out of LADs as they are activated/repressed. LADs are replicated in late S phase

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63
Q

where are active domains located in the chromosome territory

A

towards the surface and enriched in interchromosomal contacts

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64
Q

where do LADs locate in the chromosome territory

A

towards the surface but they don’t have interchromosomal contacts but are enriched in long range intrachromosomal contacts

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65
Q

where do silent domains locate in the chromosome territory

A

internal positions and are enriched in intrachromosomal contacts

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66
Q

in what phase of the cell cycle do chromosomes organise into distinct territories

A

interphase

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67
Q

describe the process of sanger sequencing

A
  • uses fluorescently labelled ddNTPs which don’t have a free 3’OH, mixed with dNTPs
  • wherever DNA pol incorporates a ddNTP it wont be able to add any other nucleotides
  • the DNA mix is run on a gel and each base is read as they separate according to size. DNA molecules are sequenced one at a time
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68
Q

what kind of sequencing was used in the human genome project

A

sanger sequencing

short regions cloned into plasmids and sequenced

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69
Q

describe the process of NGS and how it is different to sanger sequencing

A
  • DNA molecules are amplified by PCR not by cloning into individual bacteria
  • the product is spatially separated e.g. on beads, in an emulsion or on a slide
  • the DNA templates are sequenced simultaneously in a massively parallel fashion
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70
Q

in NGS how is the incorporation of specific bases detected

A

fluorescent tags
release of H ions using semiconductor chip
blockage of ions through nanopore

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71
Q

describe the solexa approach of NGS

A

bind single DNA molecules to surface
amplify
visualise clusters that form with a camera

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72
Q

describe the sequencing by synthesis solexa/illumina ngs approach

A

bind single DNA molecules to surface, amplify, visualise clusters with camera

  • add nucleotides and pol
  • image array, remove label and terminator
  • add fresh nucleotides and pol
  • sequence clusters in parallel
  • different colours –> different bases
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73
Q

how do we sequence when there is no reference genome available

A

de novo genome assembly - 35-100bp leads aligned with each other to produce a consensus genome
for this we need at least 10x coverage

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74
Q

what regions in particular are difficult to sequence

A

repetitive sequences are difficult to sequence

centromeres and telomeres are very difficult to sequence

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75
Q

how much coverage is required when sequencing to identify SNPs

A

10-30x

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76
Q

give 3 applications of NGS

A
  • identify driver mutations for cancer and personalise treatment
  • look for SNPs to study chronic disease therapy
  • RNAseq - transcriptome analysis, epigenetic analysis
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77
Q

describe the process of RNA seq

A
  1. RNA isolation from sample
  2. cDNA amplification
  3. library preparation and sequencing
  4. data analysis and alignment to reference genome
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78
Q

what is higher through put RNA seq or RT qPCR

A

RNA seq - samples processed quicker

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79
Q

which sequencing technique would be most appropriate to quantify gene expression

A

RNA seq

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80
Q

give 3 applications for RNA seq

A
  • identify up or down regulated genes using RPKM score and validate results using RT-qPCR or western blotting
  • gene ontology - are altered gens associated with particular functions
  • ingenuity pathway analysis - are altered genes associated with particular pathways
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81
Q

what is RNAi

A

regulatory mechanism of translation by small RNAs in eukaryotes

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82
Q

what is the difference between micron RNAs and small interfering RNAs

A

miRNA - inhibit mRNA translation

siRNA - induce mRNA degradation and inhibit mRNA translation

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83
Q

describe the process of RNAi function

A
  1. ds region of pre-miRNA or pre-siRNA is cut by dicer which recognises the stem structure and cleaves off the loop, releasing a 22bp RNA
  2. A ss miRNA or siRNA associates with proteins to form RISC
  3. RISC binds to cellular mRNA due to complementarity with the miRNA or siRNA within RISC
  4. siRNA has high complementarity –> mRNA degradation. miRNA has low complementarity –> no cleavage, translation inhibited
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84
Q

are miRNAs restricted to binding one mRNA

A

no - because mismatches are permitted

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85
Q

give 2 examples in biology where miRNAs are particularly important

A

development and differentiation

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86
Q

how are siRNA useful in research

A

they are used to KO proteins

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87
Q

what are some practical issues with RNAi

A

specificity - is it only targeting the gene of interest
interferon response - non specific cellular response to dsRNA
incomplete breakdown - reversible
delivery to humans - stability and quantity

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88
Q

describe the application of RNAi in Huntington’s disease

A

using an miRNA expression construct contained in an AAV vector and virus injected, mutant Htt RNA levels decreased with increasing time post infection as seen from RT qPCR normaqlised to PPIA
less Htt protein and less aggregates resulted
motor coordination and depressive phenotype improvements
miRNA bind mRNA and affects its stability and translation efficiency

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89
Q

what is Huntington’s disease and what causes it

A

it is caused by a single dominant allele
aggregation of mutant Htt results in damage to brain cells leading to gradual loss of coordination, mental ability decline and personality changes
polyglutamine tracts are toxic in some neurons and we get aggregates

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90
Q

describe the ASO clinical trial for Huntington’s

A

ASO injected intrathecally and targets an intron in the pre-mRNA of the Huntington transcript - treatment well tolerated - further trials to see if progression slows

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91
Q

what is a key difference between siRNAs and ASO

A

ASO can cross the membrane and enter the nucleus easily but siRNA can’t

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92
Q

how does the AAV virus work

A

infects dividing and non dividing cells
doesn’t replicate in humans
doesn’t integrate into genome (decrease cancer risk)
no pathogenic activity
limitation - only small DNA construct possible

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93
Q

what is CRISPR cas9

A

system of adaptive bacterial immunity used to defend against bacteriophage

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94
Q

what is cas9

A

programmable RNA guided DNA endonuclease

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95
Q

how does CRISPR cas9 work

A

we have a cas9 and a 98bp sgRNA. the protospacer RNA guides the cas9 to the corresponding sequence I the genome. cas9 cuts both strands of the genome. changing the protospacer redirects the cas9. the cleavage by cas9 leaves a ds break. a sequence of interest can then be inserted or the break is repaired naturally which can induce mutation.

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96
Q

what are the 2 mechanisms for fixing ds break from CRISPR cas9

A

non homologous end joining (NHEJ)

homology dependent repair (HDR)

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97
Q

what is the mechanism of NHEJ

A

stick ends back together, sometimes inducing mutations that result in a frameshift –> functional KO
can occur at any stage of the cell cycle
1. trimming of overhanging nucleotides to make blunt ends
2. ligase joins strands together

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98
Q

why might the HHEJ mutations be useful

A

in the lab for studying gene function but they are not useful for normal cells

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99
Q

what is the mechanism of HDR

A

can only occur in the G2 phase of the cell cycle
no errors are introduced
1. nucleolytic processing with nucleoprotein filaments attached at break to make overhang
2. search for the homologous region on the other chromosomal copy (overhang invades other copy of chromosome
3. joint molecule formation
4. strand elongation
5. base pairing with other damaged strand
6. gap filling and ligation

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100
Q

compare the stage of the cell cycle that NHEJ and HDR occur

A

NHEJ can occur at any stage of the cell cycle whereas HDR can only occur in the G2 phase

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101
Q

state 4 practical issues with CRISPRcas9

A
  • off target cleavage
  • delivery method - ex vivo vs in vivo
  • NHEJ mutations are variable
  • HDR deficient in non dividing cells and absent in terminally differentiated/non-dividing cells
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102
Q

describe the CRISPR cas9 application in Duchenne muscular dystrophy

A

exon skipping is a viable therapeutic approach as loss of some exon in the rod domain doesn’t affect protein function
they used CRISPR cas9 to cleave before and after the stop codon so its not read
delivered by AAV vector
RT-qPCR revealed edited alleles and some dystrophin was restored, improving the DMD phenotype

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103
Q

what is Duchenne muscular dystrophy

A

most severe and common type of muscular dystrophy
affects mainly males and is characterised by wasting away of muscle
caused by a range of mutations in the X linked dystrophin gene (severe phenotypes result from frameshift mutations)
death usually occurs by congestive heart failure

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104
Q

why is dystrophin important

A

it is important for muscular strength and acts like a shock absorber
loss of it makes cells fragile and muscle cells/fibres degenerate

105
Q

where in the cell is dystrophin found

A

just inside the muscle cell membrane

106
Q

what is mendelian inheritance

A

inheritance patterns that obey the law of segregation and the law of independent assortment

107
Q

what is simple mendelian inheritance

A

single gene with two different alleles that have a simple dominant/recessive relationship

108
Q

what are the F2 genotypic and phenotypic ratios for a self cross (Yy x Yy)

A

gen - 1:2:1

phen - 3:1

109
Q

what are the F2 genotypic and phenotypic ratios for a test cross (Yy x yy)

A

gen - 1:1

phen - 1:1

110
Q

give 4 examples of mendelian single gene/monogenetic diseases

A

myotonic dystrophy
sickle cell anaemia
haemophilia
Rett’s syndrome

111
Q

what are the 4 mendelian pedigree patterns

A

autosomal dominant/recessive

X linked dominant/recessive

112
Q

how is earwax a classical mendelian trait

A

a SNP in the ABCCII gene (glycine –> alanine) leads to dry earwax if homozygous, it is a recessive trait
having dry earwax in poorer climate is a selective advantage

113
Q

what is myotonic dystrophy

A

it is caused by a trinucleotide repeat downstream of the coding region in the 3’ UTR of the DMPK gene and is an autosomal dominant condition
the repeats cause the RNA to develop abnormal hairpin folds which bind splice regulating proteins, forming RNA complexes that accumulate in nuclei
this can disrupt function by altering 2 classes of RNA binding splice regulators (introns may not be removed)
muscle weakness
myotonia (difficulty relaxing muscles)
heart problems
breathing problems
digestive problems
mental problems
eye problems

114
Q

what is the myotonic dystrophy correlation between repeat size and severity

A

the longer the repeat the greater the severity (>37 repeats = unstable)

115
Q

what is the normal number of microsatellite repeats

A

5-37

116
Q

describe the importance of RNA binding splice regulators

A

loss of MBNL1 function leads to improper slicing of proteins involved in muscle contraction and growth
MBNL2 acts at a genetic level to maintain muscle fibre structure such as collagens

117
Q

what is interesting to note about the genotype and phenotype ratios in myotonic dystrophy

A

genotypic ratios follow mendels laws but the phenotypic ratios don’t

118
Q

what is anticipation and how is it displayed in myotonic dystrophy

A

phenotype gets more severe and occurs earlier in successive generations
MD - cataracts –> congenital defects

119
Q

what is somatic mosaicism

A

an individual may have cells and tissues that differ in repeat count
genetic defect may very in tissues and change over time

120
Q

what is penetrance

A

the proportion of people with a repeat expansion for MD that will actually develop symptoms
symptoms can be so mild that it is not diagnosed

121
Q

what is the range of the number of repeats in a pre symptomatic person and an affected person for MD

A

pre - 38 50

affected - 50 - 400

122
Q

what is sickle cell anaemia

A

HB surface mutation where glutamic acid (hydrophilic) –> valine (hydrophobic) - single base change
the mutant Hbs has an exposed hydrophobic region specific to the B globin
Hb molecules crystallise into a fibre with sickle shaped cells and oxygen carrying capacity is greatly reduced

123
Q

what is the structure of the Hba protein

A

it has 4 protein chains (2alpha and 2beta)in heterotetramer

124
Q

what is the phenotype of someone whos genotype is Hba/Hbs

A

less severe - sickle cell trait - incomplete dominance - haploinsufficiency (not making enough normal protein)
Hba incompletely dominant to Hbs

125
Q

what test can we do to diagnose sickle cell anaemia

A

PCR, restriction digest, gel electrophoresis
2 bands - normal
3 bands - carrier
1 band - affected
normal has the restriction site on it - mutant version does not

126
Q

how can dominance relations vary with the phenotype under consideration

A 
HB - codominance (alleles expressed at the same level)
rbc shape - sea level (Hba dominant), high altitude (incomplete dominance)
malaria susceptibility (Hbs dominant) 
presence absence of anaemia (Hba dominant)
127
Q

which 2 conditions are pleiotropic and what does it mean

A

MD and sickle cell

it means there is one gene involved but many phenotypic effects

128
Q

dominance relations affect ………….. but have no bearing on ..………….. …………….. of alleles

A

phenotype

random segregation

129
Q

name 3 X linked recessive conditions

A

haemophilia, DMD, ocular albinism

130
Q

name 3 X linked dominant conditions

A

these are often fatal

rickets, retts, fragile x syndrome

131
Q

describe the inheritance pattern of an x linked recessive trait

A

typically transferred from grandfather through carrier daughter to half of their grandsons
males are more likely to be affected
females are mosaics for mutant and normal chromosomes and normally show an intermediate phenotype (clinically unaffected but biochemically abnormal)
females can be severely affected with heavily skewed x inactivation

132
Q

describe the inheritance pattern of x linked dominant trait

A

affected males pass the condition to all of their daughters but none of their sons
affected heterozygous females crossed with unaffected males pass the condition on to half of their sons and daughters

133
Q

in x linked dominant traits male/female lethality is common

A

females are more likely to be affected because male lethality is common

134
Q

what is retts syndrome

A

neurological disorder mainly affecting girls
variable characteristics - development stalls after one year, poor speech, repetitive hand movements, poor posture
it is an x linked dominant condition caused by MECP2 (transcriptional repressor) mutation
all affected females are heterozygote and the variability stems from relative x inactivation in the brain - the more normal inactivation the more severe

135
Q

what is the lyon hypothesis

A

one X chromosome is active and the other is inactive (barr body) on body cells which equalises the activity of x linked genes in males and females

136
Q

what are barr bodies

A

inactivated chromosomes attached to the edge of the nuclear membrane

137
Q

what is XIC

A

X inactivation centre located in the long arm of X
it contains an unusual gene called XIST which expresses an non coding function 17kbp RNA molecule which is expressed only when more than one x chromosome is found in the same cell
there is a blocking factor that binds to the other X chromosome XIC to prevent its inactivation

138
Q

what is the mechanism of XIST

A

it is transcribed but nor translated and appears to act as RNAi. it binds to the x chromosome and is involved in its translocation to the nuclear periphery

  1. XIST transcription makes RNAi
  2. RNA binds to X chromosome from which it is transcribed
  3. methylation ad histone deacetylation attract proteins that form heterochromatin, inactivating the chromosome
139
Q

how does increasing the number of X chromosomes affect the level of XIST expression

A

increasing X chromosomes increases XIST expression to increase X inactivation as only one X chromosome can be expressed in a cell

140
Q

explain the colour pattern of tortoishell cats and why they cant be cloned

A

heterozygous at x linked orange gene - random x inactivation
cant be cloned because the random x inactivation occurs after the cloning process

141
Q

multigenic traits do/don’t follow predictable traits of inheritance

A

do

142
Q

what is meant by 100% penetrance

A

if you have the mutation you will get the disorder

143
Q

genes on different chromosomes are linked/inherited independently

A

inherited independently

144
Q

what is the phenotypic ratio of an F2 dihybrid self cross

A

9:3:3:1

145
Q

what is the phenotypic ratio expected in a dihybrid test cross

A

1:1:1:1

146
Q

what is epistasis

A

interaction among phenotypic effects of different genes

147
Q

when can epistasis occur

A

whenever 2 or more loci interact to create phenotypes and an allele at one locus masks or modifies the effect of the allele at other loci

148
Q

the gene hiding/being hidden is epistatic

A

the one hiding or masking the effect is epistatic

149
Q

genes with epistatic relationships tend to code for proteins that work in the same/different processes

A

same

150
Q

what is recessive epistasis and give an example

A

a recessive mutation in one gene masks the phenotypic effect of another e.g. golden retrievers - pigment not deposited in hair shaft

151
Q

what is dominant epistasis and give an example

A

dominant allele of one gene masks the action of either allele of another gene e.g. pig coat - white overrides whats happening at the E locus

152
Q

what is redundancy in phenotypes and give an example

A

don’t need both genes to make the phenotype - one can compensate for the loss of the other e.g. snapdragons - wherever a dominant allele is expressed the trait is expressed - one allele is sufficient for pigment

153
Q

what are modifier genes and give an example

A

genes that have a subtle secondary effect which alters the phenotypes produced by the primary genes e.g. mutant causes mouse tail shortening but another gene affects length - not all mutant tails are the same length

154
Q

what is meant by a modifying environment and give an example

A

the environment may influence the effect of genotype on phenotype e.g. Siamese cats - coat colour changes where temp is lower (extremities)

155
Q

describe the deafness trait and how modifier gens can have an influence

A

deafness is a homozygous recessive trait but those with a dominant modifier gene have perfect hearing

156
Q

what is polygenic inheritance and give examples

A

1 trait coded by a number of genes e.g. cancer, eczema, diabetes, Alzheimer’s

157
Q

do skin colour alleles display dominance

A

no - rather each contributing allele gives an additive effect rather than a masking effect
we get paler as we lose WT alleles
ABC - black
abc - white

158
Q

what is copy number variation

A

copies very depending on what is beneficial - results from mismatch at crossover
account for up to 5% of genome

159
Q

describe the evolution of globin families

A

duplication and mutation lead to globin genes - alpha and beta
further mutation cause transposition onto different chromosomes
further mutations lead to alpha and beta gene families

160
Q

what is decipher

A

database of CNVs associated with clinical conditions

161
Q

what methods can be used to find CNVs

A

qPCR
array comparative genomic hybridisation
FISH

162
Q

describe the process of array comparative genomic hybridisation (used to detect CNVs - not position in genome though)

A
  1. sample DNA labelled with green fluorescent dye and the control red. Both samples are applied to microarray wells with complementary sequence already bound
  2. samples hybridise to well and scanner measures fluorescence which is followed by software analysis
    - equal hybridisation - equal amounts of both samples bind
    - DNA dosage loss - more control binds
    - DNA dosage gain - more patient sample binds - patient has CNVs
163
Q

give examples of areas enriched with CNVs

A

immune system and brain development/activity

164
Q

what is the AMY1 gene

A

most common CNV found on the short arm of chromosome 1 and has different numbers of tandem repeats
encodes amylase which breaks down starch

165
Q

what process do we use for AMY1 gene mapping

A

cytogenic localisation of DNA sequences with FISH

166
Q

what about AMY1 implies tandem duplications

A

it is localised to one chromosome

167
Q

AMY1: sequence differences are large/minimal and number of copies varies minimally/largely between populations. this implies that the increase in copy number has occurred recently

A

minimal sequence difference

large variation in copy number

168
Q

higher AMY1 copy number is native to high/low starch area

A

high

if you have low CNV in high starch area you may be more at risk of obesity or diabetes

169
Q

describe FISH (used to find CNV position)

A
  1. RNA probe labelled with fluorophore before hybridisation
  2. labelled probe and target denatured
  3. combining denatured probe and target allows annealing of complementary DNA sequences
  4. fluorescence microscopy - probe fluoresces upon hybridisation and we can see where the gene of interest is
170
Q

what are tandem repeats

A

Tandem repeats occur in DNA when a pattern of one or more nucleotides is repeated and the repetitions are directly adjacent to each other.

171
Q

what is CCL3LI

A

it is a chemokine encoding multi copy gene that protects against HIV-1 in greater CNVs

172
Q

what are multifactorial traits

A

those controlled by genes and the environment e.g. height

173
Q

give examples of congenital multifactorial disorders

A 
cleft lip
hip dislocation 
heart defects
neural tube defects 
pyloric stenosis 
talipes
174
Q

give examples of adult multifactorial disorders

A 
diabetes
epilepsy
glaucoma 
hypertension 
ischaemic heart disease 
manic depression
schizophrenia
175
Q

what are characteristics of traits that are completely genetically determined

A

rare
simple genetics
unifactorial
high recurrence rate

176
Q

what are characteristics of traits that are nearly completely environmentally determined

A

common
complex genetics
multifactorial
low recurrence rate

177
Q

do multifactorial traits fit a standard mendelian inheritance pattern

A

no but they occur more frequently than expected in a family

178
Q

what is the difference between multigenic and multifactorial

A

multigenic - 2 or more genes

multifactorial - multiple factors

179
Q

why do humans show less variation than chimpanzees

A

because we have grown in size as a population very rapidly so the variation seen is still representative of the smaller population

180
Q

what is population genetics

A

the study of distributions and changes of allele frequency in a population as the population is subject to mutation, selection, gene flow and genetic drift

181
Q

what is the HW equilibrium

A

population is expected to maintain near identical alleles frequencies from one generation to the next unless an agent of change acts on it

182
Q

what 6 criteria need to be met to be in HW equilibrium

A 
large population 
random mating 
all matings fertile 
no gene flow 
no mutation 
no natural selection
183
Q

what is the equation for allele frequency

A

p +q = 1

184
Q

with each generation how many de novo polymorphism do we get

A

100-200

185
Q

give examples of what selective pressure can result from

A

competition (ecological and sexual)
predation
death/illness due to parasitic organisms/infectious disease

186
Q

give an example of natural positive selection in disease

A

sickle cell anaemia protects against malaria

187
Q

do phenotypes have a set fitness level

A

no it depends on the circumstances

188
Q

what is a selective sweep

A

when an allele becomes more common in a population as a result of positive selection

189
Q

give an example of an incomplete selective sweep

A

lactase locus

190
Q

what is artificial selection and why can it be bad

A

select for desired traits and use these individuals as parents of the next generation
often traits would be disadvantageous in the natural environment e.g. chicken legs
for crops it creates a monoculture which allows easy pathogenesis

191
Q

migration/selection is capable of changing allele frequencies more readily than migration/selection

A

migration

selection

192
Q

what happens to the gene pool on immigration

A

disproportionate quantity of certain alleles brought into a population

193
Q

what happens to the gene pool on emigration

A

departing group not representative of the population gene pool
remainder of the population may also be affected by loss of alleles

194
Q

what are the 2 causes of genetic drift

A

founder effect

bottle neck effect

195
Q

genetic drift is random/non-random

A

random

196
Q

what is the bottle neck effect

A

when an event such as a natural disaster leaves the resultant population unrepresentative of the original population
mating options decrease so the population becomes very similar
e.g. survivor was colour blind then it become more prevalent

197
Q

what is the founder effect

A

when a proportion of the population leaves or gets separated by a physical barrier the genetic structure changes to match that of the founding mothers or father e.g. Amish and ellis van crevald syndrome

198
Q

what is cystic fibrosis

A

autosomal recessive disorder that causes lung congestion and infection and malabsorption of nutrients by the pancreas

199
Q

why have CF genes persisted

A

because it provided protection against TB

200
Q

what does the CF gene encode and what is its normal and mutant function

A

it is found on chromosome 7 and encodes a chloride ion pump called CFTR
normal - moves chloride ions out of the cell
mutant - doesn’t move chlorine out so we get a sticky mucous on the outside of the cell

201
Q

75% of CF mutations are …………………………..

A

a 3bp deletion of the 508th amino acid phenylalanine

202
Q

the remaining 25% of CF mutations are …………………..

A

very variable

203
Q

what is meant by CF being oligogenic

A

small number of genes are involved

204
Q

CF has low/high penetrance

A

high

205
Q

how are modifier genes implicated in CF

A

they have roles in pulmonary and intestinal functions

206
Q

how would we find modifier genes

A

using GWAs and manhattan plots to look at association with the trait
could provide an opportunity to enhance individualised treatment

207
Q

what is the role of the EHF TF modifier of CF

A

it influences 508 deletion processing and modulates epithelial tight junctions and wound repair

208
Q

what are CF treatments

A

small molecule targeting and CRISPR cas9

209
Q

what is the route o HIV infection

A
  • CCR5 on the T cell surface is a g protein coupled receptor that normally controls migration of wbcs from the blood to inflamed tissue
  • CD4 is a receptor on T cells
  • HIV entry requires CCR5 and CD4
    1. HIV binds to CD4 via glycoprotein 120 which undergoes a conformational change and binds to CCR5
    2. Another conformational change allows HIV insertion into the cell
210
Q

what are g protein

A

a protein family off receptors that sense molecules outside the cell and activate signal transduction pathways

211
Q

what are RANTES

A

chemokines that block HIV entry
they recognise CCR5 and bind to it blocking the interaction so that HIV cant enter
problem - RANTES are no longer blocking entry in HIV areas due to mutation

212
Q

describe the variation seen in CCR5

A

32bp deletion –> non functional CCR5 –> HIV resistance
carriers protected from smallpox ( as it is also intracellular)
no CCR5 = no HIV

213
Q

describe the variation seen in the ligand gene

A

CCL3L1

  • HIV suppressive chemokine and ligand for CCR5
  • downregulates CCR5 by internalising it (like RANTES)
  • having higher CNV is advantageous
  • CNV higher in Africa - slower progression
214
Q

what do GWAS tell us

A
  • insights into modifying genes, targets etc - personalised medicine
  • population specificity
  • genetic variants vs disease relationship - association vs causation
215
Q

what are confounding factors of GWAS

A

cohort size
ethnic differences
type of case and controls
environmental factors
selection bias and recruiting the subject
varying risk genes in different populations/ethnic groups
different genotyping platforms –> variable coverage
replication of data

216
Q

what is a model organism

A

organisms used in research because they are easier to study than the organism in question
we use different organisms depending on the aspect of study

217
Q

what are key model organism characteristics

A 
sexual replication that is controllable 
cheap to maintain
lots of phenotypes 
short generation time
lots of offspring 
others working on it/standardised backgrounds 
minimal ethical concerns
218
Q

what are yeast

A

single celled eukaryotes that metabolise sugar via glycolysis to form CO2 and ethanol. they have a cell wall through which antifungals can act
they can be used to study many processes

219
Q

what are the yeast cell types

A 
a (haploid)
alpha (haploid)
a/alpha (diploid)
haploid and diploid phase are stable 
we start with diploid cell and starvation induces meiosis to create haploid cells
220
Q

what is an asci

A

4 haploid cells contained in a sac (produced by meiosis ) spores are the meiotic product and the sac is formed from the mother cell

221
Q

when do asci release cells

A

when conditions are good they release they release cells and they bud

222
Q

what must we do if we want to study haploid yeast

A

break the asci so that the cells don’t mate to form diploid cells

223
Q

for haploid/diploid genotype = phenotype

A

haploid

224
Q

what does dominance/recessiveness refer to

A

how alleles affect phenotype in a heterozygous diploid

225
Q

what determines the cell type of yeast

A

the MAT locus
MAT a - a cell
MAT alpha - alpha cell
MAT a + MAT alpha - a/alpha cell

226
Q

what are techniques of classic molecular genetics

A

introduce DNA genes
KO and CRISPR
cloning
sequencing

227
Q

what is classic alternate ploidy genetics

A

studying haploid/diploid

228
Q

yeast has highly efficient and specific …………. …………..

A

homologous recombination

229
Q

which of yeast genes have been successfully KO/inactivated

A

all 6000

230
Q

homologs have the same/different in vivo function

A

same - they encode proteins with similar sequences and are descended from common ancestors

231
Q

how are new proteins often made

A

domain shuffling

232
Q

what are the proteins of worms/animals that are no found in yeast mainly responsible for

A

cell cell communication

making the multicellular body

233
Q

do yeast and animals have homologs in disease

A

yes - e.g. cancers and metabolic diseases etc

234
Q

how can we test if homologs have the same function

A

gene should complement loss of function mutation

235
Q

give examples of synthetic biology

A 
introducing foreign DNA 
artemisinin antimalarial (from plants)
bioenergy - biofuel production
236
Q 
what are the phenotypes of 
albinism 
alkaptonuria 
phenylketonuria 
cretinism
A

loss of pigment
black urine
progressive brain dysfunction
mental retardation

237
Q

what happens in the albinism pathway

A

tyrosine is not converted to melanin because of a mutant tyrosinase enzyme
it is a recessive mutation resulting in melanin being absent from the skin, hair and eye retina

238
Q

what causes alkaptonuria

A

too much substrate
caused by a recessive mutation on chromosome 3 which encodes homogentisate 1,2 - dioxygenase
HA doesn’t get converted to MA because of this and HA which is black is expelled in the urine

239
Q

what is the result of recessive mutations in the human sodium channel

A

they cause inducible and reversible muscle paralysis triggered by specific environmental stresses e.g. cold, high/low blood K, rest after exercise
critically different point mutations cause different inducible paralysis

240
Q

what are the 2 outcomes of recessive OCA2 mutations

A

blue eyed or albinism

241
Q

can mutations in different genes cause the same phenotype

A

yes

242
Q

what is zwellweger’s syndrome

A

recessive mutations in any of the 12 genes encoding peroxisomes can cause it
unable to break down FA and unable to make myelin - muscle weakness, mental retardation and generally still born
recessive mutations in different genes can act in the same pathway

243
Q

what is allelism

A

any of several forms of a gene usually arising through a mutation
if allelic mutations are on the same gene

244
Q

what is the test used for allelism

A

complementation test otherwise known as cis-trans test

- easy, definitive, mutations must be recessive

245
Q

explain why 2 albino parents can have either all albino children or no albino children

A

genes can complement each other because it is a recessive mutation
if all the children are albino then the parents have a mutation on the same gene
if non of the children are albino then there is more than one albino gene

246
Q

what are the steps in a complementation test

A
  1. determine if mutants are recessive and only proceed if they are
  2. cross the homozygous mutants
  3. phenotype F1 progeny - if mutant the mutations don’t complement - must be on same gene. if WT mutations do complement so must be on different genes
247
Q

why cant a complementation test be used with dominant mutations

A

because the test would fail whether the mutations are on the same gene or not
the dominant mutation is always going to result in a mutant phenotype so will always seem as though there is no complementation

248
Q

what are the roles of the 4 OCA genes

A

OCA1 - tyrosine enzyme - severe
OCA2 - P protein (tyrosine helper) - mild
OCA3 - tyrosine related gene - weak
OCA4 - SLC4SA2 protein (tyrosine helper) - mild

249
Q

name another albinism like syndrome described

A

hermensky pudlak - albinism plus a bleeding problem (platelet abnormality) and storage of fat- protein compound

250
Q

describe the yeast life cycle

A

MATa and MATalpha respond to a factor secreted by the opposite mating type. a and alpha haploids cannot sporulate. they mate to form the diploid cell which undergoes mitosis and then meiosis to form the 2 haploid cells

251
Q

what is a dominance test

A

mate mutant a cell with WT alpha cell - dominant mutation in a cell if alpha/a cell is mutant

252
Q

what is a prototroph

A

can manufacture all its complex building blocks
needs C N P trace metals and vitamins in media
can grow on minimal media
WT yeast are prototrophs

253
Q

what are auxotrophs

A

need C N P trace metals vitamins and the end product of the metabolic pathway they cannot perform
cant grow on minimal medium
can grow on appropriately supplemented medium

254
Q

what is a colony

A

contains progeny derived from a single cell

255
Q

what is complete medium

A

minimal medium with every known useful additive

256
Q

for growth of a mutant do all auxotrophys need to be assessed

A

yes

257
Q

what is dropout medium

A

minimal medium with all the auxotrophs with one left out

258
Q

when will an allelism test not work

A

if genes are closely linked

259
Q

how do we assess allelism if mutations are dominant

A

cross F1s and examine F2 phenotype
- some WT –> mutations on different genes
- all mutants –> mutations on same gene
also works for recessive mutations

GMC (4 decks)

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