Gene expression and regulation Flashcards
1
Q
What is the first control of gene activity?
A
Transcription
2
Q
Which processes play a big part in transcription?
A
Transcription factors
Epigenomics
3
Q
What are transcription factors?
A
Proteins that bind to specific DNA sequences
4
Q
What do gene regulation mechanisms allow?
A
The formation of different cells depending on what genes are expressed
5
Q
Describe the selectiveness of transcription
A
Transcription is very selective
Only a particular gene or group of genes are transcribed at any one time
6
Q
How much of our genes are expressed in any given time?
A
40-50%
7
Q
Why, even though only 50% of our genes are expressed in a given time, is it said that up to 90% of the transcriptome is transcribed?
A
Because different combinations of the genes are expressed throughout the body
8
Q
What has modern genome wide analysis revealed about translation?
A
Much of the genome is transcribed into mRNA but not a lot is translated into proteins
9
Q
What are the 3 phases of transcription?
A
Initiation
Elongation
Termination
10
Q
What percentage of the genome is transcribed?
A
90%
11
Q
Examples of transcribed products
A
rRNA
tRNA
mtRNA
incRNA
piRNA
snoRNA
miRNA
mRNA
12
Q
What percentage of transcribed RNA does rRNA and tRNA encompass?
A
98%
13
Q
What 2 factors does gene expression and regulation depend on?
A
Regulatory elements
Transcription factors
14
Q
Examples of regulatory elements
A
Promoters
Enhancers
15
Q
Examples of transcription factors
A
Basal
Spacial
16
Q
What are promoters?
A
Regions of DNA necessary to initiate transcription which consists of short nucleotide sequences upstream of target genes
Binding of transcription factors and RNA polymerase allows transcription of the sequence
17
Q
Where are promoters found?
A
Upstream of their target genes
18
Q
Where does DNA methylation normally occur?
A
At promoter sites
19
Q
How are promoters different between genes
A
Number
Orientation
Distance
20
Q
What are the 4 promoters for RNA polymerase II?
A
TATA box
CAAT box
GC box
Octamer box
21
Q
How many bases are TATA boxes upstream from their target?
A
25-30 bp
22
Q
How many bases are CAAT boxes upstream from their target?
A
70-80 bp
23
Q
How many bases are GC boxes upstream from their target?
A
110 bp
24
Q
How many bases are octamer boxes upstream from their target?
A
120-130 bp
25
Describe the structure of TATA boxes
8bp sequences
Composed only of T and A pairs
26
Describe the structure of CAAT boxes
CAAT or CCAAT sequences
27
Describe the structure of GC boxes
GGGCGG
Often present in multiple copies
28
Describe the structure of octamer boxes
ATGCAT
29
What happens following mutations to TATA boxes?
A reduction in transcription
Since transcription factors can't bind
30
What happens following mutations to CAAT boxes?
A reduction in transcription
31
What happens following mutations to GC boxes?
Mutations to the DNA sequence
32
What happens following mutations to octamer boxes?
Efficiency of promoters at initiating transcription is reduced
33
What are enhancers?
DNA sequences that, upon interaction with transcription factors through bending of the DNA, increases the efficiency of initiation of transcription and transcription rate
34
How do enhancers work?
Brings all the essential components required for TF binding
35
What do enhancers respond to?
Molecules inside and outside the cell
36
Are enhancers tissue specific?
Yes
37
How does the bending of DNA occur?
Transcription factors bind to the enhancers by at least 20 different proteins to form a complex that changes the configuration of the chromatin
Causes the DNA to fold, bend or loop
38
What is the advantage of the DNA looping?
Brings the distal enhancers close to the promoter site to form activated transcription complexes
This leads to activation of transcription, which increases the rate of RNA synthesis
39
What are transcription factors?
Proteins essential for initiation of transcription which are not part of the RNA polymerase molecules
Carry out the transcription process
40
What do transcription factors help RNA polymerases do?
Bind enzymes to the DNA template
Initiate and maintain transcription
Control the rate of gene expression
41
What are the two functional domains of transcription factors?
DNA binding domain
Transcriptional activating domain
42
What is the role of the DNA binding domain of transcription factors?
Binds to DNA sequences present in regulatory regions like promoters
43
What is the role of the transcriptional activating domain?
Activate transcription via protein-protein interactions with RNA polymerase
44
What is gene expression analysis?
The study of the way genes are transcribed to synthesize functional gene products
Compare the expression of genes across multiple tissues
45
What are used in gene expression analysis as reference genes?
Housekeeper genes
46
What are housekeeper genes?
They represent constitutive genes that are required for the maintenance of basic cellular function
Express in all cells of an organism in both pathological and healthy states
47
Examples of housekeeper genes used as references in gene expression analysis
ACTB
B2M
GAPDH
48
What are northern blots?
Old-school analysis technique used to look at the gene expression levels of a target gene through measuring the total RNA belonging to the gene
49
What is the difference between a control and reference gene?
Reference genes are used to check the amount of cell material analysed are consistent across samples
Control genes are samples with no RNA, just the nucleotides and enzymes used in the process of obtaining RNA in order to ensure there is no contamination
50
What do the results of northern blot indicate?
The amount of radioactivity bound in one lane is compared to see if there is overexpression or underexpression of the target gene in the cells/individuals tested
51
Describe the process of northern blot
1. Extract total RNA from a homogenized tissue sample or cell
2. Eukaryotic mRNA is isolated through oligo chromatography to isolate RNAs with polyA tails
3. RNA samples are separated by size during gel electrophoresis
4. The RNA samples are transferred to a nylon membrane
5. Once transferred onto the membrane, the RNA is immobilized to the membrane through covalent by UV light or heat
6. A radioactively labelled probe is added to the membrane, which hybridizes with the RNA strands
7. The membrane is washed to ensure that the probe has bound specifically and to prevent background signals from arising
52
What detects the hybrid signals sent from the RNA probes in Northern Blot?
X-ray film
53
What quantifies the signals sent from RNA probes in Northern Blot?
Densitometry
54
What is the main aim of PT-PCR?
Converting an RNA template into a cDNA using reverse transcriptase and PCR primers
cDNA is then used as a template for exponential amplification using PCR
55
How can we differentiate between cDNA and contaminated mRNA or DNA?
Through their weight
cDNA is much lighter than DNA because cDNA lack the introns
56
Why are forward and reverse primers required when differentiating between cDNA and mRNA?
mRNA and cDNA are so similar, that just using forward primers would not be enough to differentiate between them
57
How is Q-RT-PCR different from RT-PCR?
This is the quantitiative form of the method described above
So RT-PCR just describes the amplification, whereas Q-RT-PCR includes the quantitative measurement of the amplified DNA
58
What does Q-RT-PCR measure?
The expression of genes
By measuring the increase in fluorescent signal from DNA-binding dyes or radioactive probes
59
How can we modify cDNA to cause replication to emit fluorescent signals?
Fluorescently labeled probes can be inserted between two primers
At each round of PCR, the probe is released, giving a signal
60
What prevents the fluorescently labelled probe from fluorescing all the time during Q-RT-PCR?
A quencher
61
What is the advantage of Q-RT-PCR over northern blot?
Highly specific
Faster since you don't need gels
Safer since does not use carcinogens
62
What is the disadvantage of Q-RT-PCR over northern blot?
Expensive
63
Describe the 3 phases of Q-RT-PCR measurements
The first phase shows exponential growth of the DNA sequences - abundance of reagents
The second phase is linear, the reagents are starting to run out
The third phase indicates a plateau, as the reagents have run out and the PCR stops
64
What phase of the PCR graph is used for protein DNA quantification?
The first phase showing exponential growth
65
What does the blue line on a Q-RT-PCR graph represent?
Threshold fluorescence required to detect the signal from the PCR product
66
What does the position of the different lines on the x-axis of a Q-RT-PCR graph represent?
The more right you are, the less RNA the patient translates, since more cycles are required for the PCR product to reach threshold
67
What is SYBR-green?
An alternative way of measuring the quantity of DNA to Q-RT-PCR
68
Describe how SYBR-green works
The green dye incorporates to the double stranded DNA in the positions with cytosine
Only gives a fluorescent signal when incorporated to the double stranded DNA
The amount of fluorescence is then measured at each cycle
69
What is the advantage of SYBR-green compared to Q-RT-PCR?
Cheap
70
What is the disadvantage of SYBR-green compared to Q-RT-PCR?
Less sentive
Less specific
71
What determines the level of detail of microarrays?
The probes used
If probes bind to different areas of the gene = levels of different transcripts can be examined
If the probes bind to poly-A tails, then we can only compare the expression of a single gene variant
72
What are the disadvantages of microarrays?
Fixed content
Hard to compare across species
73
What are the advantages of microarrays?
Allows the analysis of a large number of exons
Robust and cheap technology
74
Describe the results of microarrays
The DNA from control and experimental samples are labelled with different fluorescent tags
The single stranded labelled DNA binds to complementary probes
The relative increase or decrease in DNA expression between control and experimental sample allows us to determine the mutation underlying the disease
75
What information can RNA sequencing reveal?
Novel transcripts
Splicing variants
Profile expression levels
76
How is RNA sequencing better than microarrays?
Higher resolution
Better at discriminating at very low or very high levels of expression
77
What is necessary for a differential gene expression analysis to be carried out?
Genes need to be expressed in significantly different quantities in distinct groups of the sample
78
Examples of groups undergoing differential gene expression analysis
Drug treatment vs control
Disease vs healthy
Different tissues
79
Are microarrays examples univariate or multivariate analysis?
Univariate
80
Is RNA sequencing an example of univariate or multivariate analysis?
Univriate
81
Describe how differential gene expression analysis is carried out
t-tests or ANOVA are used to look at the significance of the difference in expression between the two groups
The p-value is logged (the smaller = the more significant = the higher up on the y-axis)
The magnitude of change is plotted on the x-axis
This is done to look at whether the genes are over or underexpressed, and to what extent
82
Graphs used to show the gene expression profile of genes between the two groups
Volcano plots
Heatmaps
Single gene comparisons
83
What is important regarding gene expression studies?
We have to interpret the results obtained through gene expression studies and discover how these relate to biological function
84
Do the top genes in differential gene expression analysis always mean something?
No
85
What are ways to biologically interpret genes differentially expressed between control and test groups?
Pathway analysis
Gene set enrichment analysis
86
What is pathway analysis?
Used to identify related proteins within a pathway or building a pathway de novo from the proteins of interest
Done by associating the function of a protein using the molecular pathway that becomes faulty upon the protein mutating
87
What is gene set enrichment analysis?
Method to identify classes of genes or proteins that are over-represented in a large set of genes or proteins, and may have an association with disease phenotypes
88
What are the 3 aspects of a gene in its role in cells?
Molecular function - catalytic, calcium ion binding
Biological process - immune response
Cellular component - nucleus, mitochondrion
89
What is a functional annotation chart?
Tries to associate genes with their molecular function, biological process and cellular component
90
What are the two main clinical applications of transcriptomics in the clinic?
Disease treamtent
Patient stratification
91
How can transcriptomics help in disease treatment?
Can help in the development of targeted therapies against genes that are upregulated/downregulated
92
How can transcriptomics help in patient stratification?
Can help determine the likelihood of recurrence of a certain cancer
Help in the prognosis of a cancer patient
Predict the likelihood of developing a disease
93
Example of a transcriptomic test used to predict the likelihood of developing a disease
Corus CAD test
Looks at the likelihood of developing obstructive coronary artery disease
94
What, apart from causing DNA looping, is another function of enhancers?
Allows RNA polymerase to bind to DNA and move along the chromosome until it reaches a promoter site
