Flashcards in Analysis of Gene Expression Deck (43)
What is differential gene expression?
Different cells expressing different genes
Development of a single cell into a complex organism depends on formation of different cell types
Genes are expressed at different levels
What is differential gene expression caused by?
It is caused by changes in expression of an unchanging set of genes
Cells make selective use of their genes – they turn expression on or off depending on cues
This is controlled at many stages - most common control point is transcription - by regulatory proteins
Production of more RNA = more active protein
What do regulatory proteins do?
Genes have long control regions often >10,000 bp
These bind regulatory proteins - enhancers/repressors
The proteins bind to short stretches of DNA
They work synergistically to amplify transcription and therefore expression
Different cells have different regulatory proteins
These affect RNA polymerase binding and transcription
How can we measure gene expression?
We can look at abundance of mRNA or protein levels
We do this in more than one cell type to compare the genome
What are some methods of detection of expression looking at abundance of mRNA?
Northern blot analysis
In situ hybridization
They all rely on nucleic acid hybridisation (driven by Watson-Crick base pairings)
We know the sequence of the gene so we can design a complementary probe
Detection of expression looking at abundance of mRNA: describe northern blot analysis?
Harvest cell types
RNAs separated by SDS-PAGE
RNAs are transferred to a filter
A labelled probe hybridises to the mRNA
The probe is detected and quantified
This is a very direct method - can be quantative
The level of darkness of a band indicates more material that it could bind to - therefore more expression
This is quantified by a machine measuring the density of material there
Detection of expression looking at abundance of mRNA: describe quantitative PCR?
Harvest RNA from different cell types
Reverse transcription of mRNA into cDNA
Quantify PCR amplification with either fluorescent primers or dye that binds dsDNA
Rate of product appearance relates to concentration of mRNA
Benefits: quantitative, rapid and can detect several targets in one tube
Detection of expression looking at abundance of mRNA: describe micro arrays?
DNA oligo representing 1000s of genes are immobilised on chip
Cell mRNA copied to cDNA using reverse transcriptase and then labelled with red or green dye
cDNAs hybridised washed and scanned
Red = expression in A
Green = expression in B
Yellow = both A and B
Can detect and quantify thousands of transcripts simultaneously
Detection of expression looking at abundance of mRNA: describe RNA sequencing?
Determine abundance of all RNAs in a cell
Harvest total RNA
Perform RNA sequencing
Compare expression levels of all genes
We can look at every single gene expressed in an organism - but not cheap
Detection of expression looking at abundance of mRNA: describe In situ hybridisation?
Very different method:
Tissue is prepared by fixing and permeabilization
Addition of labelled DNA or RNA probe (fluorescently tagged)
Probe detection by microscopy
Can simultaneously show abundance of transcript expression in all tissues of an organism
No need to separate out all tissues of an organism – they can remain in situ
Reveals information of both mRNA abundance and location
What are some methods of detection of expression looking at abundance of protein?
2D gels - MS
Detection of expression looking at abundance of proteins: describe 2D gels followed by mass spectrometry?
Isolate specific cell types
Lyse cells - release proteins
Separate proteins on 2D protein gel
They move to their isoelectric point (pH where their charge becomes 0)
The gel had a fixed pH gradient
Then separate on another gel via size
Stain the separated proteins - Coomassie blue dye
They form a pattern of spots
We can compare spot patterns - possible to identify differences in protein expression
Identify by mass spectrometry - orbitrap
Detection of expression looking at abundance of proteins: describe specific antiboidies?
Isolate specific cell types
Lyse cells - release proteins
Separate proteins on a protein gel
Transfer proteins to a membrane
Probe membrane with an antibody
Detect and quantify the antibody
What can we use for protein expression detection is there is no easy marker?
If we can't detect the expression we could add a reporter gene
This will produce reporter mRNA and therefore a reporter protein
Common regulator genes - GFP, B-galactosidase, B-glucuronidase and luciferase
Where reporter protein is detected, the gene is being expressed
How is can we find how gene expression regulated?
Identify the gene regulatory sequences
Identify gene regulatory proteins
Give an example of a gene we can identify the gene regulatory sequence and the gene regulatory proteins?
Even skipped gene (Eve)
Eve is essential for development of Drosophila
It helps define formation of the segmented body plan
Acts very early in the organization of the embryo
Eve expression occurs in 7 discrete stripes
Stretches for 20 kb; >7 kb upstream and >13 kb downstream
5 regulatory sequence modules control expression in 7 stripes
Stripe modules exert control of Eve expression by interacting with over 20 regulatory proteins
The 480 nt stope 2 module - binds 4 regulatory proteins: hunchback+, bicoid+, Kruppel- and Giant-
To determine this - Eve ORF was substituted for a reporter ORF to see where the gene was expressed
How do we identify the gene regulatory sequences
Make deletions through: restriction enzyme digestion
Site directed mutagenesis
Describe identification of gene regulatory sequences through restriction enzyme digestion?
Sequence entire regulatory region
Generate restriction map
Remove sequences by double restriction enzyme digests
Introduce the altered genes into Drosophilia eggs
Removal of BstEII - BssHII fragment abolished stripe 2 expression
These 480 nts contain all signals needed for stripe 2 expression
Delete all other sequences from the gene regulatory regions
Describe identification of gene regulatory sequences through site directed mutagenesis and gene synthesis?
Site directed mutagenesis:
Can make precise nucleotide changes to define required sequences:
Precisely shorten region
Remove internal sequences
Make single/multiple nt changes
You can make any sequence you want but it is very expensive
Using these methods alone or in combination allows the regulatory sequences to be defined
How do we identify the strip 2 (eve) gene regulatory proteins?
Electrophoretic mobility-shift analysis
Describe identification of gene regulatory proteins through electrophoretic mobility-shift analysis?
1. dsDNA fragment (20-35 bp) containing a protein binding site is prepared (end-labelled)
2. A DNA probe is incubated with a protein fraction - protein-DNA complexes form
A non-specific competitor is also added to eliminate non-specific interactions
3. Run in gel electrophoresis - under native conditions to free the bound probe
4. The gel is dried and position of the probe is detected using X-ray film
The probe + cell fraction won't move as far as the probe alone on the gel
Antibody can also be used to find out the presence of a protein
Due to the extra mass of the antibody (slower) this produces a super shift on the gel
If no antibodies are available, use MS analysis
It is very simple and highly sensitive
Describe identification of gene regulatory proteins through affinity chromatography?
1. Make a DNA fragment of the regulatory region
2. Attach to a solid matrix - eg agarose
3. Add cell lysate to column -> regulatory proteins bind the immobilized DNA
This will be washed and eluted with salt
4. Analyse by MS ID protein
How can we determine where regulatory proteins bind exactly?
2 main methods:
DNA footprint analysis
Chromatin immunoprecipitation (ChIP) analysis
Describe identification of location of gene regulatory proteins through DNA footprint analysis?
This allows us to identify nucleotides that are in contact with a DNA binding protein
Purify the binding protein
Incubate the DNA with the binding protein - forms a 'hot' regulatory region
The binding protein is in excess of the probe
Add DNase I - this binds to the minor groove and produces random nicks (only cuts once on a strand)
As this produces random cutes this can result in gaps/bands varying in intensity
The binding protein will protect its own binding site
Wash away the binding protein
Look at the sizes of the remaining DNA fragments
The products are separated in electrophoresis to reveal the 'footprint'
There is a gap in the sizes of the labelled DNA fragments
The gap is where the binding protein binds
We can generate binding curves and equilibrium constants from this data
Describe identification of location of gene regulatory proteins through Chromatin immuno-precipitation (ChIP)?
Allows in vivo identification of DNA sequences associated with proteins (DNA is in native chromatin state)
Treat in vivo cells with formaldehyde or UV to cross-link - creating a temporary physical bond between DNA-protein and chromatin-protein complexes
Chromatin fragmentation - cleave DNA into 300 bp fragments by sonication, restriction endonucleases or micrococcal nuclease
Immunoprecipitation - of the protein of interest and its associated chromatin fragments - using a specific antibody
This antibody needs to be compatible - even after cross-linking, which could adversely affect it
DNA capture/isolation - antibody-chromatin complexes are collected using beads containing proteins or secondary antibodies
A series of wash steps reduces non-specific interactions
Isolate - heat (remove cross-links), digest antibody/chromatin proteins with protease K and purify the DNA with affinity chromatography
Compare fragment sequences - deduce binding protein binding site
Do this through an qPCR and then alignment
What can we conclude about Eve after these identification experiments?
This allows us to understand how Eve expression is regulated in terms of inducers and repressors
Activators bound = repressors can't bind = Eve expressed
Repressors bound = activators can't bind = Eve not expressed
This is found in strip 2 for Eve as neither repressor is activated/bound
How do we determine gene function?
We produce mutants where gene function has been perturbed
Forward genetics (Classical genetics)
First - look for a phenotype
Second - look for a genetic change
Slow laborious costly
Second - look for a phenotype
Rapid and precise
If a gene can be linked to a phenotype then this can reveal clues about its function
Describe reverse genetics?
This can specifically perturb gene expression in two ways:
Genetic - associated with irreversible changes to the genome
E.g. nt/gene deletion, promotor deletion, splicing mutant or poly(A) mutant
This perturbs expression - some might not even produce mRNA
Epigenetic - No changes to the genome (changes elsewhere)
E.g. Regulatory proteins or RNA interference
What were some older strategies that were used for genetic/epigenetic changes in organisms?
They used to rely on introduction of altered genes and homologous recombination to swap genetic material
There are drawbacks in mutational efficiency and specificity
Transposons - jump into the area you want to change (could go to the wrong place)
Insert copies into a fused egg (single cell - pronucleus)
Recombination can be unpredictable
If diploid - must replace both WT genes
Efficiency is low and cost is very high