Block 1 - genetics Flashcards
1
Q
what factors regulate gene expression
A
TFs
chromatin and histone modification
nuclear organisation
2
Q
what do ES differentiate into in the presence of low retinoic acid
A
cardiomyocytes
3
Q
what do ES differentiate into in the presence of high retinoic acid `
A
neuronal cells
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
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
6
Q
what are the steps in RT qPCR
A
- culture cells and treat them (e.g. retinoic acid)
- purify RNA from cells
- 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.
- PCR using cDNA
- fluorescent dye binds to DAN and fluoresces - fluorescence is measured and doubles with every PCR cycle
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
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
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
10
Q
what is the Ct value
A
PCR cycle number where the fluorescence becomes greater than the threshold
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
12
Q
give 3 examples of reference genes
A
B-actin
gadph
BIII-tubulin
13
Q
describe the 3 main stages in transcription
A
- initiation - polymerase binds to the promoter, forming a TF bubble near the start site. RAN polymerase catalyses phosphodiester linkage of 2 initial rNTPs
- elongation - RNA pol advances 3’ –> 5’, melting the DNA duplex and adding rNTPs to the forming RNA (forming in 5’ –>3’ direction)
- termination - at the stop site the RAN pol releases completed RNA and dissociates form DNA
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%
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
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
17
Q
TFs are modular and often act as ………..
They have ….. domains called …….
A
dimers
2
activation domain and DNA binding domain
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
19
Q
what morphological change can happen to DNA when TFs bind
A
it can bend
20
Q
how are TFs recruited
A
the TATA box recruits TFIID which recruits other basal or general TFs
21
Q
what does TFIID consist of
A
TATA binding protein (TBP) and TBP associated factors (TAF)
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
23
Q
what is the pre-initiation complex made of
A
TFs
mediator complex (coactivator)
mRNA
24
Q
how is the pre-initiation complex formed
A
- sequence specific and basal TFs recruit coactivators e.g. the mediator complex which is required for nearly all RNA pol II transcribed genes
- the mediator complex acts as a bridge between TFs, basal TFs and the c-terminal domain of RNA pol II
25
describe 3 ways that TF activity can be regulated
- conformation alteration through ligand binding/phosphorylation (alter ability to bind DNA, interact with proteins, nuclear localisation)
- expression level alteration
- alter DNA binding site accessibility
26
how is the ELK1 TF activated
phosphorylation
27
how is the CREB TF activated
ligand activation
28
what are MAPK pathways and what do they drive
mitogen activated protein kinase pathways
| - they drive proliferation or stress responses
29
what are the 3 basic steps of an MAPK pathway
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
30
what happens when MAPK are constitutively activated and where might this be seen
transcription is constitutively active so growth is continuous. this is often seen in oncogenic cancer mutations
31
what is mitogen
growth factor
32
EGF signalling leads to ……. phosphorylation and …. ……..…. ultimately leading to the formation of the PIC and transcription
ELK1
| DNA binding
33
how do we produce antibody
we inject an adjuvant bound antigen into host animal and harvest antibodies from blood after immune response has been generated
34
what is SDS
an anionic detergent used to denature protein samples and coat them in a negative charge
35
on what basis are proteins separated in sds page
based on size
36
the sds page gel is vertical/horizontal
vertical
37
in a Coomassie stain of an sds gel highly expressed proteins have thicker/narrower bands
thicker
38
describe the process of western blotting
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
39
what do western blots show
size of the band recognised
Ab specificity
relative amounts of proteins in samples
quality of protein in sample
40
what is GADPH
a loading control
41
what is immunohistochemistry and how quantitative is it
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
42
describe the steps in DNA folding
```
double helix
beads on a string (nucleosomes)
chromatin fibre of packed nucleosomes
topologically associated domains
interphase chromosome
metaphase chromosome
```
43
what is a nucleosome
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
44
what are histone tails
they contact linker DNA and other nucleosomes and are important in chromatin folding and cofactor recruitment
45
in what 2 ways does chromatin regulate gene expression
folding controlled - (access to DNA)
| platform for a range of post translational modifications - (accessibility and TF/RNApol recruitment)
46
what are the 3 core histone tail post translational modifications
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 HAT (using acetyl coA) which are recruited by TFs. we see hyperacetylation of histone n-terminal tails
49
describe histone tail deacetylation
inhibits transcription
| occurs on lysine residues by HDAC which are recruited by repressive TFs and we see deacetylation in the TF vicinity
50
what are acetylated residues bound by (what is the reader)
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
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
56
what reader binds methylated lysine residues
chromodomain containing proteins - can be associated with activation or repression
57
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
58
describe 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 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
euchromatin
61
where are rRNA genes transcribed
nucleolus
62
what are lamin associated domains
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
63
where are active domains located in the chromosome territory
towards the surface and enriched in interchromosomal contacts
64
where do LADs locate in the chromosome territory
towards the surface but they don't have interchromosomal contacts but are enriched in long range intrachromosomal contacts
65
where do silent domains locate in the chromosome territory
internal positions and are enriched in intrachromosomal contacts
66
in what phase of the cell cycle do chromosomes organise into distinct territories
interphase
67
describe the process of sanger sequencing
- 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
68
what kind of sequencing was used in the human genome project
sanger sequencing
| short regions cloned into plasmids and sequenced
69
describe the process of NGS and how it is different to sanger sequencing
- 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
70
in NGS how is the incorporation of specific bases detected
fluorescent tags
release of H ions using semiconductor chip
blockage of ions through nanopore
71
describe the solexa approach of NGS
bind single DNA molecules to surface
amplify
visualise clusters that form with a camera
72
describe the sequencing by synthesis solexa/illumina ngs approach
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
73
how do we sequence when there is no reference genome available
de novo genome assembly - 35-100bp leads aligned with each other to produce a consensus genome
for this we need at least 10x coverage
74
what regions in particular are difficult to sequence
repetitive sequences are difficult to sequence
| centromeres and telomeres are very difficult to sequence
75
how much coverage is required when sequencing to identify SNPs
10-30x
76
give 3 applications of NGS
- identify driver mutations for cancer and personalise treatment
- look for SNPs to study chronic disease therapy
- RNAseq - transcriptome analysis, epigenetic analysis
77
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
78
what is higher through put RNA seq or RT qPCR
RNA seq - samples processed quicker
79
which sequencing technique would be most appropriate to quantify gene expression
RNA seq
80
give 3 applications for RNA seq
- 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
81
what is RNAi
regulatory mechanism of translation by small RNAs in eukaryotes
82
what is the difference between micron RNAs and small interfering RNAs
miRNA - inhibit mRNA translation
| siRNA - induce mRNA degradation and inhibit mRNA translation
83
describe the process of RNAi function
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
84
are miRNAs restricted to binding one mRNA
no - because mismatches are permitted
85
give 2 examples in biology where miRNAs are particularly important
development and differentiation
86
how are siRNA useful in research
they are used to KO proteins
87
what are some practical issues with RNAi
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
88
describe the application of RNAi in Huntington's disease
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
89
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
90
describe the ASO clinical trial for Huntington's
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
91
what is a key difference between siRNAs and ASO
ASO can cross the membrane and enter the nucleus easily but siRNA can't
92
how does the AAV virus work
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
93
what is CRISPR cas9
system of adaptive bacterial immunity used to defend against bacteriophage
94
what is cas9
programmable RNA guided DNA endonuclease
95
how does CRISPR cas9 work
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.
96
what are the 2 mechanisms for fixing ds break from CRISPR cas9
non homologous end joining (NHEJ)
| homology dependent repair (HDR)
97
what is the mechanism of NHEJ
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
98
why might the HHEJ mutations be useful
in the lab for studying gene function but they are not useful for normal cells
99
what is the mechanism of HDR
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
100
compare the stage of the cell cycle that NHEJ and HDR occur
NHEJ can occur at any stage of the cell cycle whereas HDR can only occur in the G2 phase
101
state 4 practical issues with CRISPRcas9
- 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
102
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 AAV vector
RT-qPCR revealed edited alleles and some dystrophin was restored, improving the DMD phenotype
103
what is Duchenne muscular dystrophy
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
104
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
105
where in the cell is dystrophin found
just inside the muscle cell membrane
106
what is mendelian inheritance
inheritance patterns that obey the law of segregation and the law of independent assortment
107
what is simple mendelian inheritance
single gene with two different alleles that have a simple dominant/recessive relationship
108
what are the F2 genotypic and phenotypic ratios for a self cross (Yy x Yy)
gen - 1:2:1
| phen - 3:1
109
what are the F2 genotypic and phenotypic ratios for a test cross (Yy x yy)
gen - 1:1
| phen - 1:1
110
give 4 examples of mendelian single gene/monogenetic diseases
myotonic dystrophy
sickle cell anaemia
haemophilia
Rett's syndrome
111
what are the 4 mendelian pedigree patterns
autosomal dominant/recessive
| X linked dominant/recessive
112
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
113
what is myotonic dystrophy
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
what is the myotonic dystrophy correlation between repeat size and severity
the longer the repeat the greater the severity (>37 repeats = unstable)
115
what is the normal number of microsatellite repeats
5-37
116
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
117
what is interesting to note about the genotype and phenotype ratios in myotonic dystrophy
genotypic ratios follow mendels laws but the phenotypic ratios don't
118
what is anticipation and how is it displayed in myotonic dystrophy
phenotype gets more severe and occurs earlier in successive generations
MD - cataracts --> congenital defects
119
what is somatic mosaicism
an individual may have cells and tissues that differ in repeat count
genetic defect may very in tissues and change over time
120
what is penetrance
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
what is the range of the number of repeats in a pre symptomatic person and an affected person for MD
pre - 38 50
| affected - 50 - 400
122
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 B globin
Hb molecules crystallise into a fibre with sickle shaped cells and oxygen carrying capacity is greatly reduced
123
what is the structure of the Hba protein
it has 4 protein chains (2alpha and 2beta)in heterotetramer
124
what is the phenotype of someone whos genotype is Hba/Hbs
less severe - sickle cell trait - incomplete dominance - haploinsufficiency (not making enough normal protein)
Hba incompletely dominant to Hbs
125
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
126
how can dominance relations vary with the phenotype under consideration
```
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
which 2 conditions are pleiotropic and what does it mean
MD and sickle cell
| it means there is one gene involved but many phenotypic effects
128
dominance relations affect ………….. but have no bearing on ..………….. …………….. of alleles
phenotype
| random segregation
129
name 3 X linked recessive conditions
haemophilia, DMD, ocular albinism
130
name 3 X linked dominant conditions
these are often fatal
| rickets, retts, fragile x syndrome
131
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
132
describe the inheritance pattern of x linked dominant trait
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
in x linked dominant traits male/female lethality is common
females are more likely to be affected because male lethality is common
134
what is retts syndrome
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
what is the lyon hypothesis
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
what are barr bodies
inactivated chromosomes attached to the edge of the nuclear membrane
137
what is XIC
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
what is the mechanism of XIST
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
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
140
explain the colour pattern of tortoishell cats and why they cant be cloned
heterozygous at x linked orange gene - random x inactivation
cant be cloned because the random x inactivation occurs after the cloning process
141
multigenic traits do/don't follow predictable traits of inheritance
do
142
what is meant by 100% penetrance
if you have the mutation you will get the disorder
143
genes on different chromosomes are linked/inherited independently
inherited independently
144
what is the phenotypic ratio of an F2 dihybrid self cross
9:3:3:1
145
what is the phenotypic ratio expected in a dihybrid test cross
1:1:1:1
146
what is epistasis
interaction among phenotypic effects of different genes
147
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
148
the gene hiding/being hidden is epistatic
the one hiding or masking the effect is epistatic
149
genes with epistatic relationships tend to code for proteins that work in the same/different processes
same
150
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
151
what is dominant epistasis and give an example
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
what is redundancy in phenotypes and give an example
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
what are modifier genes and give an example
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
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)
155
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
156
what is polygenic inheritance and give examples
1 trait coded by a number of genes e.g. cancer, eczema, diabetes, Alzheimer's
157
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
158
what is copy number variation
copies very depending on what is beneficial - results from mismatch at crossover
account for up to 5% of genome
159
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
160
what is decipher
database of CNVs associated with clinical conditions
161
what methods can be used to find CNVs
qPCR
array comparative genomic hybridisation
FISH
162
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
163
give examples of areas enriched with CNVs
immune system and brain development/activity
164
what is the AMY1 gene
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
what process do we use for AMY1 gene mapping
cytogenic localisation of DNA sequences with FISH
166
what about AMY1 implies tandem duplications
it is localised to one chromosome
167
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
minimal sequence difference
| large variation in copy number
168
higher AMY1 copy number is native to high/low starch area
high
| if you have low CNV in high starch area you may be more at risk of obesity or diabetes
169
describe FISH (used to find CNV position)
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
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.
171
what is CCL3LI
it is a chemokine encoding multi copy gene that protects against HIV-1 in greater CNVs
172
what are multifactorial traits
those controlled by genes and the environment e.g. height
173
give examples of congenital multifactorial disorders
```
cleft lip
hip dislocation
heart defects
neural tube defects
pyloric stenosis
talipes
```
174
give examples of adult multifactorial disorders
```
diabetes
epilepsy
glaucoma
hypertension
ischaemic heart disease
manic depression
schizophrenia
```
175
what are characteristics of traits that are completely genetically determined
rare
simple genetics
unifactorial
high recurrence rate
176
what are characteristics of traits that are nearly completely environmentally determined
common
complex genetics
multifactorial
low recurrence rate
177
do multifactorial traits fit a standard mendelian inheritance pattern
no but they occur more frequently than expected in a family
178
what is the difference between multigenic and multifactorial
multigenic - 2 or more genes
| multifactorial - multiple factors
179
why do humans show less variation than chimpanzees
because we have grown in size as a population very rapidly so the variation seen is still representative of the smaller population
180
what is population genetics
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
what is the HW equilibrium
population is expected to maintain near identical alleles frequencies from one generation to the next unless an agent of change acts on it
182
what 6 criteria need to be met to be in HW equilibrium
```
large population
random mating
all matings fertile
no gene flow
no mutation
no natural selection
```
183
what is the equation for allele frequency
p +q = 1
184
with each generation how many de novo polymorphism do we get
100-200
185
give examples of what selective pressure can result from
competition (ecological and sexual)
predation
death/illness due to parasitic organisms/infectious disease
186
give an example of natural positive selection in disease
sickle cell anaemia protects against malaria
187
do phenotypes have a set fitness level
no it depends on the circumstances
188
what is a selective sweep
when an allele becomes more common in a population as a result of positive selection
189
give an example of an incomplete selective sweep
lactase locus
190
what is artificial selection and why can it be bad
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
migration/selection is capable of changing allele frequencies more readily than migration/selection
migration
| selection
192
what happens to the gene pool on immigration
disproportionate quantity of certain alleles brought into a population
193
what happens to the gene pool on emigration
departing group not representative of the population gene pool
remainder of the population may also be affected by loss of alleles
194
what are the 2 causes of genetic drift
founder effect
| bottle neck effect
195
genetic drift is random/non-random
random
196
what is the bottle neck effect
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
what is the founder effect
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
what is cystic fibrosis
autosomal recessive disorder that causes lung congestion and infection and malabsorption of nutrients by the pancreas
199
why have CF genes persisted
because it provided protection against TB
200
what does the CF gene encode and what is its normal and mutant function
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
75% of CF mutations are …………………………..
a 3bp deletion of the 508th amino acid phenylalanine
202
the remaining 25% of CF mutations are …………………..
very variable
203
what is meant by CF being oligogenic
small number of genes are involved
204
CF has low/high penetrance
high
205
how are modifier genes implicated in CF
they have roles in pulmonary and intestinal functions
206
how would we find modifier genes
using GWAs and manhattan plots to look at association with the trait
could provide an opportunity to enhance individualised treatment
207
what is the role of the EHF TF modifier of CF
it influences 508 deletion processing and modulates epithelial tight junctions and wound repair
208
what are CF treatments
small molecule targeting and CRISPR cas9
209
what is the route o 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
210
what are g protein
a protein family off receptors that sense molecules outside the cell and activate signal transduction pathways
211
what are RANTES
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
describe the variation seen in CCR5
32bp deletion --> non functional CCR5 --> HIV resistance
carriers protected from smallpox ( as it is also intracellular)
no CCR5 = no HIV
213
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
214
what do GWAS tell us
- insights into modifying genes, targets etc - personalised medicine
- population specificity
- genetic variants vs disease relationship - association vs causation
215
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 --> variable coverage
replication of data
216
what is a model organism
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
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
```
218
what are yeast
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
what are the yeast cell types
```
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
what is an asci
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
when do asci release cells
when conditions are good they release they release cells and they bud
222
what must we do if we want to study haploid yeast
break the asci so that the cells don't mate to form diploid cells
223
for haploid/diploid genotype = phenotype
haploid
224
what does dominance/recessiveness refer to
how alleles affect phenotype in a heterozygous diploid
225
what determines the cell type of yeast
the MAT locus
MAT a - a cell
MAT alpha - alpha cell
MAT a + MAT alpha - a/alpha cell
226
what are techniques of classic molecular genetics
introduce DNA genes
KO and CRISPR
cloning
sequencing
227
what is classic alternate ploidy genetics
studying haploid/diploid
228
yeast has highly efficient and specific …………. …………..
homologous recombination
229
which of yeast genes have been successfully KO/inactivated
all 6000
230
homologs have the same/different in vivo function
same - they encode proteins with similar sequences and are descended from common ancestors
231
how are new proteins often made
domain shuffling
232
what are the proteins of worms/animals that are no found in yeast mainly responsible for
cell cell communication
| making the multicellular body
233
do yeast and animals have homologs in disease
yes - e.g. cancers and metabolic diseases etc
234
how can we test if homologs have the same function
gene should complement loss of function mutation
235
give examples of synthetic biology
```
introducing foreign DNA
artemisinin antimalarial (from plants)
bioenergy - biofuel production
```
236
```
what are the phenotypes of
albinism
alkaptonuria
phenylketonuria
cretinism
```
loss of pigment
black urine
progressive brain dysfunction
mental retardation
237
what happens in the albinism pathway
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
what causes alkaptonuria
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
what is the result of recessive mutations in the human sodium channel
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
what are the 2 outcomes of recessive OCA2 mutations
blue eyed or albinism
241
can mutations in different genes cause the same phenotype
yes
242
what is zwellweger's syndrome
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
what is allelism
any of several forms of a gene usually arising through a mutation
if allelic mutations are on the same gene
244
what is the test used for allelism
complementation test otherwise known as cis-trans test
| - easy, definitive, mutations must be recessive
245
explain why 2 albino parents can have either all albino children or no albino children
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
what are the steps in a complementation 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 WT mutations do complement so must be on different genes
247
why cant a complementation test be used with 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
248
what are the roles of the 4 OCA genes
OCA1 - tyrosine enzyme - severe
OCA2 - P protein (tyrosine helper) - mild
OCA3 - tyrosine related gene - weak
OCA4 - SLC4SA2 protein (tyrosine helper) - mild
249
name another albinism like syndrome described
hermensky pudlak - albinism plus a bleeding problem (platelet abnormality) and storage of fat- protein compound
250
describe the yeast life cycle
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
what is a dominance test
mate mutant a cell with WT alpha cell - dominant mutation in a cell if alpha/a cell is mutant
252
what is a prototroph
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
what are auxotrophs
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
what is a colony
contains progeny derived from a single cell
255
what is complete medium
minimal medium with every known useful additive
256
for growth of a mutant do all auxotrophys need to be assessed
yes
257
what is dropout medium
minimal medium with all the auxotrophs with one left out
258
when will an allelism test not work
if genes are closely linked
259
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
