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Genomics > Gene expression and regulation > Flashcards

Gene expression and regulation Flashcards

1
Q

What is the first control of gene activity?

A

Transcription

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2
Q

Which processes play a big part in transcription?

A

Transcription factors

Epigenomics

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3
Q

What are transcription factors?

A

Proteins that bind to specific DNA sequences

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4
Q

What do gene regulation mechanisms allow?

A

The formation of different cells depending on what genes are expressed

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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

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6
Q

How much of our genes are expressed in any given time?

A

40-50%

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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

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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

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9
Q

What are the 3 phases of transcription?

A

Initiation

Elongation

Termination

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10
Q

What percentage of the genome is transcribed?

A

90%

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11
Q

Examples of transcribed products

A

rRNA

tRNA

mtRNA

incRNA

piRNA

snoRNA

miRNA

mRNA

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12
Q

What percentage of transcribed RNA does rRNA and tRNA encompass?

A

98%

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13
Q

What 2 factors does gene expression and regulation depend on?

A

Regulatory elements

Transcription factors

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14
Q

Examples of regulatory elements

A

Promoters

Enhancers

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15
Q

Examples of transcription factors

A

Basal

Spacial

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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

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17
Q

Where are promoters found?

A

Upstream of their target genes

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18
Q

Where does DNA methylation normally occur?

A

At promoter sites

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19
Q

How are promoters different between genes

A

Number

Orientation

Distance

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20
Q

What are the 4 promoters for RNA polymerase II?

A

TATA box

CAAT box

GC box

Octamer box

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21
Q

How many bases are TATA boxes upstream from their target?

A

25-30 bp

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22
Q

How many bases are CAAT boxes upstream from their target?

A

70-80 bp

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23
Q

How many bases are GC boxes upstream from their target?

A

110 bp

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24
Q

How many bases are octamer boxes upstream from their target?

A

120-130 bp

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25
Q

Describe the structure of TATA boxes

A

8bp sequences

Composed only of T and A pairs

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26
Q

Describe the structure of CAAT boxes

A

CAAT or CCAAT sequences

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27
Q

Describe the structure of GC boxes

A

GGGCGG

Often present in multiple copies

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28
Q

Describe the structure of octamer boxes

A

ATGCAT

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29
Q

What happens following mutations to TATA boxes?

A

A reduction in transcription

Since transcription factors can’t bind

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30
Q

What happens following mutations to CAAT boxes?

A

A reduction in transcription

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31
Q

What happens following mutations to GC boxes?

A

Mutations to the DNA sequence

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32
Q

What happens following mutations to octamer boxes?

A

Efficiency of promoters at initiating transcription is reduced

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33
Q

What are enhancers?

A

DNA sequences that, upon interaction with transcription factors through bending of the DNA, increases the efficiency of initiation of transcription and transcription rate

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34
Q

How do enhancers work?

A

Brings all the essential components required for TF binding

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35
Q

What do enhancers respond to?

A

Molecules inside and outside the cell

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36
Q

Are enhancers tissue specific?

A

Yes

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37
Q

How does the bending of DNA occur?

A

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

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38
Q

What is the advantage of the DNA looping?

A

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
Q

What are transcription factors?

A

Proteins essential for initiation of transcription which are not part of the RNA polymerase molecules

Carry out the transcription process

40
Q

What do transcription factors help RNA polymerases do?

A

Bind enzymes to the DNA template

Initiate and maintain transcription

Control the rate of gene expression

41
Q

What are the two functional domains of transcription factors?

A

DNA binding domain

Transcriptional activating domain

42
Q

What is the role of the DNA binding domain of transcription factors?

A

Binds to DNA sequences present in regulatory regions like promoters

43
Q

What is the role of the transcriptional activating domain?

A

Activate transcription via protein-protein interactions with RNA polymerase

44
Q

What is gene expression analysis?

A

The study of the way genes are transcribed to synthesize functional gene products

Compare the expression of genes across multiple tissues

45
Q

What are used in gene expression analysis as reference genes?

A

Housekeeper genes

46
Q

What are housekeeper genes?

A

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
Q

Examples of housekeeper genes used as references in gene expression analysis

A

ACTB

B2M

GAPDH

48
Q

What are northern blots?

A

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
Q

What is the difference between a control and reference gene?

A

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
Q

What do the results of northern blot indicate?

A

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
Q

Describe the process of northern blot

A
  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
Q

What detects the hybrid signals sent from the RNA probes in Northern Blot?

A

X-ray film

53
Q

What quantifies the signals sent from RNA probes in Northern Blot?

A

Densitometry

54
Q

What is the main aim of PT-PCR?

A

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
Q

How can we differentiate between cDNA and contaminated mRNA or DNA?

A

Through their weight

cDNA is much lighter than DNA because cDNA lack the introns

56
Q

Why are forward and reverse primers required when differentiating between cDNA and mRNA?

A

mRNA and cDNA are so similar, that just using forward primers would not be enough to differentiate between them

57
Q

How is Q-RT-PCR different from RT-PCR?

A

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
Q

What does Q-RT-PCR measure?

A

The expression of genes

By measuring the increase in fluorescent signal from DNA-binding dyes or radioactive probes

59
Q

How can we modify cDNA to cause replication to emit fluorescent signals?

A

Fluorescently labeled probes can be inserted between two primers

At each round of PCR, the probe is released, giving a signal

60
Q

What prevents the fluorescently labelled probe from fluorescing all the time during Q-RT-PCR?

A

A quencher

61
Q

What is the advantage of Q-RT-PCR over northern blot?

A

Highly specific

Faster since you don’t need gels

Safer since does not use carcinogens

62
Q

What is the disadvantage of Q-RT-PCR over northern blot?

A

Expensive

63
Q

Describe the 3 phases of Q-RT-PCR measurements

A

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
Q

What phase of the PCR graph is used for protein DNA quantification?

A

The first phase showing exponential growth

65
Q

What does the blue line on a Q-RT-PCR graph represent?

A

Threshold fluorescence required to detect the signal from the PCR product

66
Q

What does the position of the different lines on the x-axis of a Q-RT-PCR graph represent?

A

The more right you are, the less RNA the patient translates, since more cycles are required for the PCR product to reach threshold

67
Q

What is SYBR-green?

A

An alternative way of measuring the quantity of DNA to Q-RT-PCR

68
Q

Describe how SYBR-green works

A

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
Q

What is the advantage of SYBR-green compared to Q-RT-PCR?

A

Cheap

70
Q

What is the disadvantage of SYBR-green compared to Q-RT-PCR?

A

Less sentive

Less specific

71
Q

What determines the level of detail of microarrays?

A

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
Q

What are the disadvantages of microarrays?

A

Fixed content

Hard to compare across species

73
Q

What are the advantages of microarrays?

A

Allows the analysis of a large number of exons

Robust and cheap technology

74
Q

Describe the results of microarrays

A

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
Q

What information can RNA sequencing reveal?

A

Novel transcripts

Splicing variants

Profile expression levels

76
Q

How is RNA sequencing better than microarrays?

A

Higher resolution

Better at discriminating at very low or very high levels of expression

77
Q

What is necessary for a differential gene expression analysis to be carried out?

A

Genes need to be expressed in significantly different quantities in distinct groups of the sample

78
Q

Examples of groups undergoing differential gene expression analysis

A

Drug treatment vs control

Disease vs healthy

Different tissues

79
Q

Are microarrays examples univariate or multivariate analysis?

A

Univariate

80
Q

Is RNA sequencing an example of univariate or multivariate analysis?

A

Univriate

81
Q

Describe how differential gene expression analysis is carried out

A

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
Q

Graphs used to show the gene expression profile of genes between the two groups

A

Volcano plots

Heatmaps

Single gene comparisons

83
Q

What is important regarding gene expression studies?

A

We have to interpret the results obtained through gene expression studies and discover how these relate to biological function

84
Q

Do the top genes in differential gene expression analysis always mean something?

A

No

85
Q

What are ways to biologically interpret genes differentially expressed between control and test groups?

A

Pathway analysis

Gene set enrichment analysis

86
Q

What is pathway analysis?

A

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
Q

What is gene set enrichment analysis?

A

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
Q

What are the 3 aspects of a gene in its role in cells?

A

Molecular function - catalytic, calcium ion binding

Biological process - immune response

Cellular component - nucleus, mitochondrion

89
Q

What is a functional annotation chart?

A

Tries to associate genes with their molecular function, biological process and cellular component

90
Q

What are the two main clinical applications of transcriptomics in the clinic?

A

Disease treamtent

Patient stratification

91
Q

How can transcriptomics help in disease treatment?

A

Can help in the development of targeted therapies against genes that are upregulated/downregulated

92
Q

How can transcriptomics help in patient stratification?

A

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
Q

Example of a transcriptomic test used to predict the likelihood of developing a disease

A

Corus CAD test

Looks at the likelihood of developing obstructive coronary artery disease

94
Q

What, apart from causing DNA looping, is another function of enhancers?

A

Allows RNA polymerase to bind to DNA and move along the chromosome until it reaches a promoter site

Genomics (9 decks)

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