Genetics Flashcards
1
Q
What regulate gene expression?
A
- Transcription Factors
- Chromatin and Histone modicifications
- Nuclear organisation and Heterochromatin
2
Q
How can gene expression be regulated at the DNA, RNA and protein levels?
A
3
Q
What do Embryonic Stem cells differentiate into in the presence of low retinoic acid?
A
Embryonic Stem cells differentiate into cardiomyocytes in the presence of low retinoic acid.
4
Q
What do Embryonic Stem cells differentiate into in the presence of high retinoic acid?
A
Embryonic Stem cells differentiate into Neuronal cells in the presence of high retinoic acid.
5
Q
What are the marker genes for Embryonic Stem cells, Cardiomyocytes and Neurons and what are their functions?
A
Embryonic Stem Cell Genetic Marker
OCT4 - A transcription factor for self-renewal.
Cardiomyocytes
Cardiac troponin C (TNNC1) binds Ca2+ to activate muscle contraction.
Neurons
MAP2 - stabalises microtubules in dendrites.
6
Q
What is quantitative/real time PCR used for?
A
quantitative RT-PCR / real time RT-PCR
A technique for amplifying and quantifying the amount of a specific RNA present in your sample
- q = quantitative / real time
- RT = reverse transcriptase
- PCR = polymerase chain reaction
7
Q
What are the steps to quantifying mRNA expression?
A
- Culture cells and treat them according to your experimental protocol
- Purify RNA from the cells
- Reverse transcriptase reaction to synthesise cDNA from RNA
- PCR: Polymerase Chain Reaction
- Fluorescent dye binds to DNA and fluoresces - fluorescence is measured and doubles with every cycle of PCR.
8
Q
How is RNA converted into cDNA for PCR?
A
Reverse Transcriptase is used to synthesise cDNA from RNA as RNA cannot be used in PCR. mRNA has poly A tail so oligo(dT) or random hexamer primers are used. The primers anneal to the poly A tail and Reverse Transcriptase forms the cDNA which is then separated from the template RNA using alkali.
9
Q
What are the steps involved in PCR?
A
10
Q
What materials are required for RT qPCR?
A
- Reverse Transcriptase
- Oligo(dT) or random hexamer primers
- Gene and reference gene
- dNTPs
- buffer
- High quality RNA
- PCR mix
- Dye - SYBR
- Fluorescence measuring machinery and computer for analysis
- Taq polymerase
11
Q
What is semi-quantitative PCR?
A
PCR is said to be Semi-quantitative when Agarose gel electrophoresis is conducted after a set number of cycles. Detected by DNA stain.
12
Q
How does the fluorescence work in RT q-PCR?
A
SYBR green fluorescent dye intercalates into double stranded DNA products.
13
Q
What is the Ct value?
A
The Ct value is the PCR cycle number at which fluorescence becomes greater than the threshold.
14
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.
15
Q
Give 3 examples of reference genes
A
- β-actin
- Gapdh
- βIII-tubulin
16
Q
Describe the main stages of transcription
A
Initiation
- Polymerase binds to promoter sequence in duplex DNA. “Closed complex”.
- Polymerase melts duplex DNA near transcription start site, forming a trasncription bubble. “Open complex”.
- Polymerase catalyses phosphodiester linkage of two initial rNTPs.
Elongation
- Polymerase advances 3’ → 5’ down template strand, melting duplex DNA and adding rNTPs to growing RNA.
Termination
- At trascription stop site, polymerase releases completed RNA and dissociates from DNA.
17
Q
What are the 3 types of RNA?
A
- ribosomal RNAs (rRNA), RNA polymerase I 80% of RNA
- messenger RNAs (mRNA), RNA polymerase II 5% of RNA
- transfer RNAs (tRNA), RNA polymerase III 15% of RNA
18
Q
What is RNA pol II transcription initiation regulated by?
A
Transcription Factors that bind to specific DNA sequences and recruit coactivators and RNA polymerase II.
19
Q
Name and describe 3 Gene regulatory elements
A
- Promoter proximal element - 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
20
Q
Transcription Factors are modular and often act as ……
They have ….. domains called …….
A
Transcription Factors are modular and often act as dimers
They have 2 domains called activation domain (AD) and DNA binding domain.
21
Q
What is the serum response factor?
A
It is a Transcription Factor 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.
22
Q
What morphological change can happen to DNA when Transcription Factors bind?
A
The morphological changes that can happen to DNA is that it can bend.
23
Q
How are Transcription Factors recruited?
A
The TATA box recruits TFIID, which consists of TATA-binding protein (TBP) and TBP- associated factors (TAFs).
24
Q
What does TFIID consist of?
A
TATA binding protein (TBP) and TBP associated factors (TAFs).
25
What is the role of the pre-initiation complex?
It positions RNA polymerase at the transcription start site and allows for phosphorylation of the polymerase C-terminal domain of RNA polymerase II.
26
What is the pre-initiation complex made of?
* Transcription Factors
* Mediator complex (coactivator)
* mRNA
27
How is the pre-initiation complex formed?
1. Sequence specific and basal Transcription Factors recruit coactivators e.g. the mediator complex which is required for nearly all RNA polymerase II transcribed genes.
2. The mediator complex acts as a bridge between Transcription Factors, basal Transcription Factors and the C-terminal domain of RNA polymerase II.
28
Describe 3 ways that Transcription Factor activity can be regulated.
1.
29
How is the ELK1 TF activated?
Phosphorylation-activated DNA binding.
30
How is the CREB Transcription Factor activated?
Ligand-activated DNA binding
31
What is the MAPK pathway?
1. Growth factors bind to cell surface receptors
2. Receptor activation sets up cascades of protein phosphorylation inside the cell, amplifying the signal.
3. At the bottom of the cascade, transcription factors get phosphorylated and activated, driving transcription.
* MAPK signalling can drive proliferation, or stress responses
* Oncogenic mutations that constitutively activate MAPK signalling are common in cancer
32
What is Mitogen?
Mitogen is a growth hormone.
33
EGF signalling leads to ……. phosphorylation and ..…. ultimately leading to the formation of the PIC and transcription
EGF signalling leads to Elk1 phosphorylation and DNA binding, ultimately leading to the formation of the PIC and transcription.
34
How are antibodies produced?
1. Antigen: purified protein or short peptide
2. Antigen is fused to an adjuvant protein, which stumulates an immune reaction, and is injected into host animal (e.g. rabbit, mouse, goat)
3. Host generates an immune response to the protein, and blood samples are taken a regular intervals (e.g. once a month) to see if the serum now contains antibodies to the protein of interest.
35
What is SDS-PAGE and how does it work?
SDS-PAGE is Sodium dodecyl sulphate - polyacrylamide gel electrophoresis
1. Protein samples denatured with the anionic detergent
2. SDS Proteins migrate towards the positive anode
36
How are proteins differentiated using SDS-PAGE?
Proteins are differentiated between on the basis of their respective molecular weights (kD).
37
In a Coomassie stain of a SDS-PAGE gel, highly expressed proteins have **thicker**/**narrower** bands
## Footnote
**_Select the correct description_**
In a Coomassie stain of a SDS-PAGE gel, highly expressed proteins have **thicker** bands.
38
What are the steps involved in Western Blotting?
_Western Blotting_
1. Conduct SDS-PAGE on proteins.
2. Transfer proteins to membrane using electric current (blotting)
3. Incubate membrane with Antibody specific for protein being studied (usually raised in rabbit or mouse)
4. Secondary Antibody bound to Horse Radish Peroxide enzyme is added which recognises the primary Antibody and amplifies the signal.
5. HRP substrate added and light emitted is detected by CCD camera
39
What does Western Blotting reveal?
_Western Blots can show:_
* Size of band recognised by antibody
* Specificity of antibody
* Relative amounts of protein in samples
* Quality of protein sample
40
What is GAPDH?
GADPH is a loading control that shows the same amount of protein in each lane (every band will have the same thickness).
41
What is immunohistochemistry and how quantitative is it?
It provides spatial information and uses Antibodies to detect proteins in cultured cells or tissues. Often visualised using secondary Antibodies with fluorescent tags.
It is semi quantitative.
42
Describe the steps in DNA folding
1. double helix
2. beads on a string (nucleosomes)
3. chromatin fibre of packed nucleosomes
4. topologically associated domains
5. interphase chromosome
6. metaphase chromosome
43
What is the nucleosome?
The nucleosome is an octamer of core histone proteins: H2A, H2B, H3, H4 (2 of each) 146 bp of DNA is wrapped twice around the octamer.
44
What is the role of Histone tails?
* Contact linker DNA and other nucleosomes
* Are important for chromatin folding and co-factor recruitment
45
What are the two oppurtunities chromatin provide to regulate gene expression?
1. The folding of the chromatin can be regulated, thus controlling the access of proteins to the DNA.
2. The chromatin provides a platform for a range of posttranslational modifications that control DNA accessibility and transcription factor and RNA polymerase recruitment.
46
What are some Post-translational modification of core histone 'tails'?
* Acetylation
* Phosphorylation
* Methylation
47
Why is histone tail modification of significance?
The modifications bind cofactors which cause activation/folding/repression.
48
Describe histone tail acetylation
* Promotes transcription
* Occurs on lysine residues by Histone Acetyl Transferase (using acetyl coA) which are recruited by Transcription Factors. We see hyperacetylation of histone n-terminal tails.
49
Describe histone tail deacetylation
Inhibits transcription
Occurs on lysine residues by Histone Deacetylase which are recruited by repressive Transcription Factors and we see deacetylation in the Transcription Factor vicinity.
50
What are acetylated residues bound by?
Bromodomain-containing proteins (high affinity where multiple acetylated sites exist in proximity). Coactivators with bromodomains promote binding of other Trancription Factors and the mediator complex leading to RNA polymerase II recruitment and pre-initiation complex formation.
51
What is constitutive heterochromatin?
Chromatin that is always highly condensed into heterochromatin
52
Where is constitutive heterochromatin found?
Centromeres and telomeres
53
What is DNA methylation?
Works with repressive histone modifications to condense and silence chromatin.
54
Describe histone tail methylation
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.
55
Describe histone tail demethylation.
KDM removes methyl groups from lysine residues and turn them into lysine once again.
56
What reader binds methylated lysine residues?
Chromodomain containing proteins can be associated with activation or repression
57
Where is Constitutive heterochromatin found?
Constitutive heterochromatin is found at centromeres and telomeres.
58
What are characteritistics of Constitutive Heterochromatin?
Highly condensed, repressive histone modifications, methylated DNA, no meiotic recombination, replicated in late S phase, non-coding RNA important for centromeric chromatin.
59
Describe characteriatics of facultative heterochromatin
Condensed, inactive genes, repressive histone modifications, methylated DNA (where silenced), replicated in later S phase, non-coding RNA may be involved in repressive regions.
60
If genes are active is the chromatin in the form of **euchromatin** or **heterochromatin**?
If genes are active the chromatin is in the form of **euchromatin**.
61
What are centromeres?
Region where spindle fibres attach and pull apart chromatids during mitosis. They are repetitive sequences; their chromatin contains specialised histones. The chromatin are also always very highly condensed into heterochromatin.
62
What are telomeres?
They are essentially chromasome ends. They consist of DNA repeats, maintained during replication by telomerase. The chromatin structure is always very highly condensed into heterochromatin. Telomere length decreases as an organism ages, except in stem cells.
63
Where are rRNA genes transcribed?
rRNA genes are transcribed in the nucleolus.
64
Where are Lamin Associated Domains located?
Lamin-associated domains (LAD) are located at the nuclear periphery.
65
What are Lamin-associated Domains?
* Heterochromatic regions of chromosomes
* Associated with lamins of the nuclear membrane
* Most genes in LADs are silenced
* LADs are replicated late in S phase
* Genes can move in and out of LADs as they are activated or repres
66
Where are active domains located in the chromosome territory?
Towards the surface and enriched in interchromosomal contacts.
67
Where do LADs locate in the chromosome territory?
Lamin - associated domains locate towards the surface but they don't have interchromosomal contacts but are enriched in long range intrachromosomal contacts.
68
Where do silent domains locate in the chromosome territory?
Internal positions and are enriched in intrachromosomal contacts
69
Describe euchromatin
Less condensed, active genes, gene promoters have active histone modifications, gene promoters are not methylated, replication occurs throughout the S phase, acetylated histones
70
In what phase of the cell cycle do chromosomes organise into distinct territories?
Chromosomes organise into distinct territories in interphase.
71
Describe the process of sanger sequencing
* Uses fluorescently labelled dNTPs which don't have a free 3'OH, mixed with dNTPs
* Wherever DNA polymerase incorporates a dNTP 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
72
What kind of sequencing was used in the human genome project?
Sanger sequencing was used in the human genome project. Short DNA regions were cloned into plasmids and sequenced accordingly.
73
Describe the process of Next Generation Sequencing/High throughput sequencing and how it is different to sanger sequencing
* DNA molecules within the library amplified by PCR, not by cloning individually into bacteria
* Amplified DNA templates are spatially segregated: eg on beads, in an emulsion, or on a slide.
* DNA templates are sequenced simultaneously in a massively parallel fashion
74
In Next Generation Sequencing how is the incorporation of specific bases detected?
Incorporation of specific bases can be detected in various ways, eg:
* By fluorescent tags using a camera
* By release of H ions using a semiconductor chip
* By how they block the flow of ions through a nanopore
75
Describe the solexa approach of Next Generation Sequencing
76
Describe the sequencing by synthesis Solexa/Ilumina Next Generation Sequencing approach
1. Bind single DNA molecules to surface, amplify, visualise clusters with camera
2. Add nucleotides and DNA poylmerase
3. Image array, remove label and terminator
4. Add fresh nucleotides and DNA polymerase
5. Sequence clusters in parallel
Different colours → Different bases
77
How do we sequence when there is no reference genome available?
* 35 – 100 bp reads are aligned with each other to produce a consensus genome sequence.
* Need at least 10x coverage. Repetitive regions are harder to sequence.
78
What regions in particular are difficult to sequence in De novo genome assembly?
Repetitive sequences are difficult to sequence. Thus, centromeres and telomeres are very difficult to sequence.
79
How much coverage is required when sequencing to identify SNPs?
10-30x coverage is needed to identify SNPs.
80
Give 3 applications of Next Generation Sequencing
* Identify driver mutations for cancer and personalise treatment
* Look for SNPs to study chronic disease therapy
* RNAseq - transcriptome analysis, epigenetic analysis
81
Describe the process of RNA seq
1. RNA isolation from sample
2. cDNA amplification
3. Library preparation and sequencing
4. Data analysis and alignment to reference genome
82
What sequencing method has a higher throughput? **RNA-seq** or **RT-qPCR**?
**RNA seq** has a higher throughput as the samples are processed quicker.
83
Which sequencing technique would be most appropriate to quantify gene expression?
RNA seq is the most appropriate DNA sequencing method to quantify gene expression.
84
Give 3 applications of RNA-seq data
* 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 e.g. MAPK signalling, Insulin signalling etc.
85
What is the RPKM score?
RPKM score: Reads Per Kilobase of transcript per Million mapped reads
86
What is RNAi?
Regulatory mechanism of translation by small RNAs in eukaryotes.
87
What is the difference between micron RNAs and small interfering RNAs?
* miRNA inhibit mRNA translation
* siRNA induce mRNA degradation and inhibit mRNA translation
88
Describe the process of RNAi function
1. double stranded 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 single stranded 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
89
Are miRNAs restricted to binding one mRNA?
90
What are miRNAs important for?
miRNAs are important for development and differentiation.
91
How are siRNA useful in research?
siRNA are used to knockout proteins in research.
92
What are some practical issues with RNA interference?
* **Specificity** – is siRNA only targeting the gene of interest?
* **Interferon response** – a non-specific cellular response to dsRNA Need to use negative control siRNA, and repeat with different siRNA triggers.
* **Incomplete knockdown**; Reversible
* **Delivery** in humans – how to stably get the siRNA to the correct location?
93
Describe the application of RNAi in Huntington's disease
* Using an miRNA expression construct contained in an Adeno-Aociated Virus vector and virus injected, mutant Htt RNA levels decreased with increasing time post infection as seen from RT-qPCR normalised 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
94
What is Huntington's disease and what causes it?
* 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
95
Decribe the Phase I Clinical trial of IONIS-HTTRx
* Antisense DNA oligonucleotide (ASO) targeting an intron in the pre-mRNA of the Huntingtin transcript. DNA-RNA hybrid digested by Rnase H
* Injected intrathecally (as ASOs do not cross the blood-brain barrier), once every 4 weeks, on 4 occasions
* The treatment was well tolerated, further trials are needed to see if progression slows.
96
What is a key difference between siRNAs and ASO?
Unlike siRNA molecules, ASOs can cross cell membranes and enter the nucleus.
97
How does the AAV virus work?
_The Adeno-Associated Virus_
* Infects dividing and non-dividing cells.
* Doesn't replicate in human cells
* Version used for gene therapy doesn't integrate into the genome (reduced cancer risk)
* No apparent pathogenic activity
* Only small DNA constructs possible.
98
What is CRISPR Cas 9?
* CRISPR: **_c_**lustered **_r_**egularly **_i_**nterspaced **_s_**hort **_p_**alindromic **_r_**epeat
* Cas: **_C_**RISPR **_as_**sociated genes
* It is a system of adaptive bacterial immunity used to defend against bacteriophage
99
What is cas9?
Programmable RNA guided DNA endonuclease.
100
How does CRISPR cas9 work?
We have a Cas9 and a 98bp sgRNA. the protospacer RNA guides the Cas9 to the corresponding sequence in the genome. Cas9 cuts both strands of the genome. Changing the protospacer redirects the Cas9. The cleavage by Cas9 leaves a double stranded break. A sequence of interest can then be inserted or the break is repaired naturally which can induce mutation.
101
What are the 2 mechanisms for fixing a double stranded break from CRISPR Cas9?
1. Non homologous end joining (NHEJ)
2. Homology dependent repair (HDR)
102
What is the mechanism of Non Homologous End Joining?
* DNA-PK bound to KU80/KU70 heterodimers. The heterodimers bind to the sites of the double stranded DNA break. In doing so, trim thwe overhanging nucleotides to make blunt ends.
* The ligase then joins the seperated DNA strands together and remove the DNA-PK and KU80/KU70 heterodimers.
103
Why might the NHEJ mutations be useful?
In the lab for studying gene function but they are not useful for normal cells.
104
What is the mechanism of Homology Directed Repair?
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
105
Compare the stages of the cell cycle that Non Homologous End Joining and Homology Directed Repair can occur in.
NHEJ can occur at any stage of the cell cycle whereas HDR can only occur in the G2 phase.
106
State 4 practical issues with CRISPR Cas9
1. Off target cleavage
2. Delivery method; ex vivo versus in vivo approaches
3. NHEJ mutations variable
4. HDR inefficient in dividing cells, absent (?) in terminally differentiated, non-dividing cells
107
Describe the CRISPR Cas9 application in Duchenne muscular dystrophy
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 Adeno-Associated Virus vector
RT-qPCR revealed edited alleles and some dystrophin was restored, improving the DMD phenotype.
108
What is Duchenne muscular dystrophy?
* DMD is the most severe and common type of muscular dystrophy Characterised by the wasting away of muscles – voluntary muscles, heart and breathing muscles.
* Caused by a range of mutations in the X-linked dystrophin gene.
109
Why is dystrophin important?
It is important for muscular strength and acts like a shock absorber
Loss of it makes cells fragile and muscle cells/fibres degenerate.
110
Where in the cell is dystrophin found?
Dystrophin is found just inside the cell membrane.
111
What is mendelian inheritance?
Inheritance patterns that obey the law of segregation and the law of independent assortment.
112
What is simple mendelian inheritance?
Simple Mendelian Inheritance is where a single gene with two different alleles that have a simple dominant/recessive relationship.
113
What are the F2 genotypic and phenotypic ratios for a self cross (Yy x Yy)?
The genotypic ratio of a self-cross is 1:2:1
The phenotypic ratio of a self cross is 3:1
114
What are the F2 genotypic and phenotypic ratios for a test cross? (Yy x yy)
* The genotypic ratio would be 1:1
* The phenotypic ratio would also be 1:1
115
Give 4 examples of Mendelian single gene/monogenetic diseases
Four examples of Mendelian single gene/monogenetic disease include:
* Myotonic dystrophy
* Sickle cell anaemia
* Haemophilia
* Rett's syndrome
116
What are four Mendelian pedigree patterns?
The four Mendelian pedigree patterns are:
* Autosomal dominant/recessive
* X linked dominant/recessive
117
How is earwax a classical Mendelian trait?
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
118
What causes Myotonic Dystrophy?
* It is caused by a trinucleotide repeat downstream of the coding region in the 3' untranslated region 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)
119
What are symptoms of Myotonic Dystophy?
* Muscle weakness
* Myotonia (difficulty relaxing muscles)
* Heart problems
* Breathing problems
* Digestive problems
* Mental problems
* Eye problems
120
What is the Myotonic Dystrophy correlation between repeat size and severity?
The longer the repeat the greater the severity (\>37 repeats = unstable)
121
What is the normal number of microsatellite repeats?
The normal number of microsatellite repeats are 5-37.
122
Describe the importance of RNA binding splice regulators
* 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.
123
What is interesting to note about the genotype and phenotype ratios in Myotonic Dystrophy?
The genotypic ratios follow mendels laws however, the phenotypic ratios don't.
124
What is anticipation and how is it displayed in Myotonic Dystrophy?
* Anticipation in genetics is where as a genetic disorder is passed on to the next generation, the symptoms of the genetic disorder become apparent at an earlier age with each generation. In most cases, an increase in the severity of symptoms is also noted.
* In Myotonic Dystophy, a phenotype such as cataracts can eventually become worsened in later generations and become evident as congenital defects.
125
What is Somatic Mosaicism?
* Somatic mosaicism refers to the occurrence of two genetically distinct populations of cells within an individual, derived from a postzygotic mutation.
* In contrast to inherited mutations, somatic mosaic mutations may affect only a portion of the body and are not transmitted to progeny.
126
What is penetrance?
* The proportion of people with a repeat expansion for Myotonic Dystrophy that will actually develop symptoms.
* Symptoms can be so mild that it is not diagnosed.
127
What is the range of the number of repeats in a pre symptomatic person and an affected person for Myotonic Dystrophy?
* The range of the number of repeats in a pre-symptomatic person is 38 - 50
* The range of the number of repeats in an affected person is 50 - 400
128
What is Sickle Cell Anaemia?
Hb surface mutation where glutamic acid (hydrophilic) --\> valine (hydrophobic) - single base change
The mutant HbS has an exposed hydrophobic region specific to the Beta globin
Hb molecules crystallise into a fibre with sickle shaped cells and oxygen carrying capacity is greatly reduced.
129
What is the structure of the Hba protein?
It has 4 protein chains (2 alpha and 2 beta) in heterotetramer
130
What is the phenotype of someone whho has a genotype of Hba/Hbs
* The symptoms would be less severe as the sickle cell trait would exhibit incomplete dominance leading to haploinsufficiency (not making enough normal protein)
* Hba incompletely dominant to Hbs
131
What test can we do to diagnose sickle cell anaemia?
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.
132
How can dominance relations vary with the phenotype under consideration?
* Hb - codominance (alleles expressed at the same level)
* red blood cell shape - sea level (Hba dominant),
* high altitude (incomplete dominance)
* malaria susceptibility (Hbs dominant)
* presence absence of anaemia (Hba dominant)
133
Which two conditions are pleiotropic and what does pleiotropic mean?
* Myotonic Dystrophy and sickle cell
* When a condition is described as pleiotropic it essentially means there is one gene involved which has many phenotypic effects.
134
Dominance relations affect ____ but have no bearing on _____ \_\_\_\_\_ of alelles.
Dominance relations affect phenotype but have no bearing on the random segregation of alleles.
135
Name 3 X-linked recessive conditions
An example of 3 X-linked conditions include:
* Haemophilia
* Duchenne Muscular Dystrophy (DMD)
* Ocular Albinism
136
Name 3 X-linked dominant conditions
* Rickets, Retts, Fragile X Syndrome
* These are often fatal conditons.
137
Describe the inheritance pattern of an X-linked recessive trait
* 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
138
* 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
139
In X-linked dominant traits **male**/**female** lethality is common
## Footnote
**_Select the correct option_**
In X-linked dominant traits **male** lethality is common
140
What is Retts Sydnrome?
* Retts syndrome is 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
141
What is the Lyon hypothesis?
One X chromosome is active and the other is inactive (barr body) on somatic cells which equalises the activity of x linked genes in males and females.
142
What are Barr bodies?
Barr bodies are inactivated chromosomes attwched to the edge of the nuclear membrane.
143
What does XIC stand for?
* **XIC** stands for **X Inactivation Centre** located in the long arm of the X chromosome.
* 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
144
What is the mechanism of XIST?
* It is transcribed but not 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
145
How does increasing the number of X chromosomes affect the level of XIST expression?
Increasing X chromosomes increases XIST expression to increase X inactivation as only one X chromosome can be expressed in a cell.
146
Explain the colour pattern of tortoiseshell cats and why they cant be cloned.
* They are heterozygous at x linked orange gene - random x inactivation
* Can't be cloned because the random x inactivation occurs after the cloning process
147
Multigenic traits **do**/**don't** follow predictable traits of inheritance.
**_Select the correct option_**
Multigenic traits **do** follow predictable traits of inheritance.
148
What is meant by 100% penetrance?
100% penetrance means that if you have the mutation you will certainly get the disorder.
149
Genes on different chromosomes are **linked**/**inherited independently**.
**_Select the correct option_**
Genes on different chromosomes are **inherited independently**.
150
What is the phenotypic ratio of an F2 dihybrid self cross?
The phenotypic ratio of an F2 dihybrid self cross is
9:3:3:1
151
What is the phenotypic ratio expected in a dihybrid test cross?
The phenotypic ratio of a dihybrid test cross is 1:1:1:1.
152
What is epistasis?
Epistasis is the interaction among phenotypic effects of different genes.
153
When can epistasis occur?
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.
154
The gene **hiding/****being hidden** is epistatic.
**_Select the correct option_**
The gene **hiding** the effect of the other gene is epistatic.
155
Genes with epistatic relationships tend to code for proteins that work in the **same**/**different** processes.
Genes with epistatic relationships tend to code for proteins that work in the **same** processes.
156
What is recessive epistasis and give an example.
A recessive mutation in one gene masks the phenotypic effect of another e.g. golden retrievers - pigment not deposited in hair shaft
157
What is dominant epistasis and give an example.
Dominant epistasis is where the 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
158
What is redundancy in phenotypes and give an example?
Redundancy in phenotypes is when you 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.
159
What are modifier genes and give an example.
Modifier genes are 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.
160
What is meant by a modifying environment and give an example.
The environment may influence the effect of genotype on phenotype e.g. Siamese cats - coat colour changes where temp is lower (extremities)
161
Describe the deafness trait and how modifier gens can have an influence
Deafness is a homozygous recessive trait but those with a dominant modifier gene have perfect hearing.
162
What is polygenic inheritance and give examples.
1 trait coded by a number of genes e.g. cancer, eczema, diabetes, Alzheimer's.
163
Do skin colour alleles display dominance?
* 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
164
What is Copy Number Variation?
* Copies vary depending on what is beneficial - results from mismatch at crossover
* Account for up to 5% of genome
165
Describe the evolution of globin families.
* 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
166
What is decipher?
Decipher is a database of CNVs associated with clinical conditions.
167
What methods can be used to find CNVs?
Methods that can be used to find CNVs include:
* qPCR
* Array comparative genomic hybridisation
* Fluorescence in situ hybridization (FISH)
168
Describe the process of array comparative genomic hybridisation (used to detect CNVs - not position in genome though).
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
169
Give examples of areas enriched with CNVs
* Immune system
* Brain development/activity
170
What is the AMY1 gene?
The AMY1 gene is the most common CNV found on the short arm of chromosome 1 and has different numbers of tandem repeats. It encodes amylase which breaks down starch.
171
What process do we use for AMY1 gene mapping?
The process used for AMY1 gene mapping is cytogenic localisation of DNA sequences with FISH.
172
What is it about AMY1 that implies tandem duplications?
AMY1 is localised to one chromosome.
173
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
## Footnote
**_Select the correct options_**
AMY1: sequence differences are **minimal** and number of copies varies **largely** between populations. this implies that the increase in copy number has occurred recently
174
Higher AMY1 copy number is native to **high**/**low** starch area.
Higher AMY1 copy number is native to **high** starch area.
* If you have low CNV in high starch area you may be more at risk of obesity or diabetes.
175
Describe Fluorescence in situ hybridization
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.
176
What are Tandem repeats?
Tandem repeats occur in DNA when a pattern of one or more nucleotides is repeated and the repetitions are directly adjacent to each other.
177
What is CCL3LI?
It is a chemokine encoding multi copy gene that protects against HIV-1 in greater CNVs.
178
What are multifactorial traits?
These are traits controlled by genes and the environment.
179
Give examples of congenital multifactorial disorders.
* Cleft lip
* Hip dislocation
* Heart defects
* Neural tube defects
* Pyloric stenosis
* Talipes
180
Give examples of adult multifactorial disorders.
* Diabetes
* Epilepsy
* Glaucoma
* Hypertension
* Ischaemic heart disease
* Manic depression
* Schizophrenia
181
What are characteristics of traits that are completely genetically determined?
* Rare
* Simple genetics
* Unifactorial
* High recurrence rate
182
What are characteristics of traits that are nearly completely environmentally determined?
* Common
* Complex genetics
* Multifactorial
* Low recurrence rate
183
Do multifactorial traits fit a standard mendelian inheritance pattern?
No but they occur more frequently than expected in a family.
184
What is the difference between multigenic trait and a multifactorial trait?
* Multigenic - 2 or more genes
* Multifactorial - multiple factors
185
Why do humans show less variation than chimpanzees?
This is because we have grown in size as a population very rapidly so the variation seen is still representative of the smaller population.
186
What is population genetics?
Population genetics is 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.
187
What is the Hardy-Weinberg Equilibrium?
The Hardy-Weinberg quilibrium states the population is expected to maintain near identical alleles frequencies from one generation to the next unless an agent of change acts on it.
188
What 6 criteria need to be met in the Hardy-Weinberg equilibrium?
the 6 criteria that need to be met in the Hardy-Weinberg Equilibrium include:
1. Large population
2. Random mating
3. All matings fertile
4. No gene flow
5. No mutation
6. No natural selection
189
What are the equations for Hardy-Weinberg Equilibrium?
* p*2 + 2*pq* + *q*2 = 1
* p + q = 1*
190
With each generation how many de novo polymorphisms do we get?
With each generation around 100-200 de novo polymorphisms are generated.
191
Give examples of what selective pressure can result from.
* Competition (ecological and sexual)
* Predation
* Death/illness due to parasitic organisms/infectious disease
192
Give an example of natural positive selection in disease
An example of natural positive selection pressure is sickle cell anaemia protecting against malaria.
193
Do phenotypes have a set fitness level?
Phenotypes do not have a set fitness level it depends on the circumstances.
194
What is a selective sweep?
A selective sweep is when an allele becomes more common in a population as a result of positive selection.
195
Give an example of an incomplete selective sweep.
An example of an incomplete selective sweep is in the lactase locus.
196
What is artificial selection and why can it be bad?
Artificial Selection is where humans:
* 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
197
**Migration**/**selection** is capable of changing allele frequencies more readily than **migration**/**selection**.
## Footnote
**_Select the correct options._**
**Migration** is capable of changing allele frequencies more readily than **selection**.
198
What happens to the gene pool on immigration?
A disproportionate quantity of certain alleles brought into a population.
199
What happens to the gene pool on emigration?
The departing group is not representative of the population gene pool. The remainder of the population may also be affected by loss of alleles.
200
What are the 2 causes of genetic drift?
The 2 causes of genetic drift include:
1. The founder effect
2. The bottle neck effect
201
Genetic drift is **random**/**non-random**.
## Footnote
**_Select the correct option._**
Genetic drift is **random**.
202
What is the bottle neck effect?
* The bottle neck effect is 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
203
What is the founder effect?
The founder effect is 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.
204
What is Cystic Fibrosis?
Cystic Fibrosis is an autosomal recessive disorder that causes lung congestion and infection and malabsorption of nutrients by the pancreas.
205
Why have CF genes persisted?
Cystic Fibrosis has persisted because it provided protection against Tuberclerosis.
206
What does the Cystic fibrosis gene encode for and what is its normal and mutant function?
* It is found on chromosome 7 and encodes a chloride ion pump called CFTR
* Wild type - 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
207
75% of Cystic Fibrosis mutations are \_\_\_\_\_
75% of Cystic Fibrosis mutations are a 3bp deletion of the 508th amino acid phenylalanine.
208
The remaining 25% of CF mutations are \_\_\_\_\_\_
The remaining 25% of CF mutations are very variable.
209
What is meant by Cystic Fibrosis being Oligogenic?
Cystic Fibrosis being Oligogenic means a small number of genes are involved.
210
Cystic Fibrosis has a **high**/**low** penetrance
**_Select the correct option_**
Cystic Fibrosis has **high** penetrance
211
How are modifier genes implicated in Cystic Fibrosis?
Modifier genes have roles in pulmonary and intestinal functions.
212
How would we find modifier genes?
We would find modifer genes using GWAs and manhattan plots to look at association with the trait. This could provide an opportunity to enhance individualised treatment.
213
What is the role of the Ets homologous factor TF modifier of Cystic Fibrosis ?
It influences 508 deletion processing and modulates epithelial tight junctions and wound repair.
214
What are Cystic Fibrosis treatments?
Treatments for Cystic Fibrosis include:
* Small molecule targeting
* CRISPR cas9
215
What is the route of HIV infection?
* 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
216
What is a G - protein?
A protein family off receptors that sense molecules outside the cell and activate signal transduction pathways.
217
What are RANTES?
* Chemokines that block HIV entry
* They recognise CCR5 and bind to it blocking the interaction so that HIV cant enter
* The issue with RANTES is that they are no longer blocking entry in HIV areas due to mutation
218
Describe the variation seen in CCR5
32bp deletion leads to a non functional CCR5. This offers the trait of HIV resistance
Carriers are also protected from smallpox as it is intracellular)
no CCR5 = no HIV
219
Describe the variation seen in the ligand gene.
_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
220
What does GWAS tell us?
GWAS allows for:
* Insights into modifying genes, targets etc - personalised medicine
* Population specificity
* Genetic variants vs disease relationship - association vs causation
221
What are confounding factors of GWAS?
* 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 leading to variable coverage
* Replication of data
222
What is a model organism?
Model organisms are 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.
223
What are key model organism characteristics?
* 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
224
What is yeast?
* Yeast are 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
225
What are the yeast cell tyoes?
* 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
226
What is an asci?
An asci are 4 haploid cells contained in a sac (produced by meiosis ) spores are the meiotic product and the sac is formed from the mother cell.
227
When do asci release cells?
Asci release cells when conditions are good and release the cells which then begin to bud.
228
What must we do if we want to study haploid yeast?
We must break the asci so that the cells don't mate to form diploid cells.
229
For **haploid**/**diploid** genotype = phenotype
**_Select the correct option_**
For **haploid** genotype = phenotype
230
What does dominance/recessiveness refer to?
Dominance/Recessiveness refers to how alleles affect phenotype in a heteroygous diploid.
231
What determines the cell type of yeast?
The MAT locus determines the cell type of yeast.
* MAT a - a cell
* MAT alpha - alpha cell
* MAT a + MAT alpha - a/alpha cell
232
What are techniques of classic molecular genetics?
Techniques of classical molecular genetics include:
* Introduce DNA genes
* KO and CRISPR
* Cloning
* Sequencing
233
What is classic alternate ploidy genetics?
Classic alternate ploidy genetics is the study of haploid/diploid cells.
234
Yeast has highly effecient and specific ____ \_\_\_\_\_
Yeast has highly efficient and specific homologous recombination.
235
Which of yeast genes have been successfully KO/inactivated?
All 6000 yeast genes have been knocked out/inactivated.
236
Homologs have the **same**/**different** in vivo function.
* Homologs have the **same** in vivo function.
* They encode proteins with similar sequences and are descended from common ancestors.
237
How are new proteins often made?
new proteins are often made through domain shuffling.
238
What are the proteins of worms/animals that are not found in yeast mainly reThe nsponsible for?
The proteins of animals/worms that are not found in yeast are mainly responsible for making the multicellular body and cell communication.
239
Do yeast and animals have homologs in disease?
Humans and yeast have homologs in disease such as cancers and metabolic diseases.
240
How can we test if homologs have the same function?
The way to test if homologs have the same function the gene should complement the loss of function mutation.
241
Give examples of synthetic biology.
Examples of synthetic biology include:
* Introducing foreign DNA
* Artemisinin antimalarial (from plants)
* Bioenergy - biofuel production
242
What are the phenotypes of albinism, alkapotnuria, phenylketonuria and cretinsism?
* Loss of pigment
* Black urine
* Progressive brain dysfunction
* Mental retardation
243
What happens in the albinism pathway?
In the albanism pathway, tyrosine is not converted into melanin because of a mutant tyrosinase enzyme. It is a recessive mutation resulting in melanin being absent from the skin, hair and eye retina.
244
What causes alkapotnuria?
What causes alkapotnuria is essentially too much substrate.
it is caused by a recessive mutation on chromosome 3 which encodes for homogenitisate 1,2-dioxygenase. Homogentisic acid thus doesn't get converted into Maleyacetoacetic acid. Homogentisic acid is black so when it is then expelled in the urine, it gives urine a black appearance.
245
What is the result of recessive mutations in the human sodium channel?
The result of recessive mutations in the human sodium channel is 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.
246
What are 2 outcomes of recessive OCA2 mutastions?
The 2 outcomes of recessive OCA2 mutations are penotypes such as having blue eyes or albinism.
247
Can mutations in different genes cause the same phenotype?
Mutations in different genes can cause the same genotype.
248
What is Zwellweger's syndrome?
Zwellweger's syndrome is caused by recessive mutations in any of the 12 genes encoding peroxisomes. It can cause make it unable to break down Fatty acids and unable to make myelin.
* This can lead to:
* muscle weakness, mental retardation and generally still born
Recessive mutations in different genes can act in the same pathway
249
What is allelism?
Allelism is any of several forms of a gene usually arising through a mutation. If allelic mutations are on the same gene.
250
What is the test used for allelism?
The test for allelism is a complementation test otherwise known as cis-trans test
* It's easy, definitive, however mutations must be recessive.
251
Explain why 2 albino parents can have either all albino children or no albino children.
Albino parents can either have no albino children or all abino children. This is because 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
252
What are the steps in the completion test?
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 Wild Type mutations do complement so must be on different genes
253
Why can't a comepletion test be used with dominant mutations?
* A completion test can't be used on dominant mutations 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.
254
What are the roles of the 4 OCA genes?
OCA1 - tyrosine enzyme - severe albinism
OCA2 - P protein (tyrosine helper) - mild albinism
OCA3 - tyrosine related gene - weak albinism
OCA4 - SLC4SA2 protein (tyrosine helper) - mild albinism
255
Name another albinism like syndrome described.
Hermensky pudlak - albinism plus a bleeding problem (platelet abnormality) and storage of fat- protein compound.
256
Describe the yeast life cycle.
1. Essentially, MATa and MATalpha respond to a factor secreted by the opposite mating type.
2. a and alpha haploids cannot sporulate.
3. They mate to form the diploid cell which undergoes mitosis
4. Then meiosis to form the 2 haploid cells
257
What is a phototroph?
A phototroph can manufacture all its complex building blocks.
* It needs C N P trace metals and vitamins in media.
* It can grow on minimal media
* Wild type yeast are phototrophs.
258
What are autotrophs?
Autotrophs need 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
259
What is a colony?
A colony contains progeny derived from a single cell.
260
What is a complex medium?
A complex medium is a minimal medium with every known useful additive.
261
For growth of a mutant do all autotrophs need to be assessed?
For growth of a mutant all autotrophs need to be assessed.
262
What is dropout medium?
Dropout medium is the minimal medium with all the autotrophs with one left out.
263
When will an allelism test not work?
If genes are closely linked the allelism tests won't work.
264
How do we assess allelism if mutations are dominant?
* Cross F1s and examine F2 phenotype
* Some WT --\> mutations on different genes
* All mutants --\> mutations on same gene
* also works for recessive mutations
265
