genetics lecture 2 - regulation of transcription 2 Flashcards
what are the stages of antibody production?
1) Antigen: Purified protein or short peptide
2) Antigen is injected into host animal (e.g. rabbit, mouse, goat) along with an adjuvant to stimulate an immune reaction.
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
what is an SDS-PAGE (Sodium dodecyl sulphate- polyacrylamide gel electrophoresis)?
Protein samples denatured with the anionic detergent SDS
Proteins migrate towards the positive anode
Proteins separated on the basis of size
Coomassie blue stain for all proteins
what is western blotting?
what can western blotting show?
size of band recognised by antibody
specificity of antibody
relative amounts of protein in samples
quality of protein sample
increase in expression of the transcription factor GATA4 during cardiomyocyte differentiation
what is Immunofluorescence and examples?
Use antibodies to detect proteins in cultured cells or tissue sections
Visualise using a secondary antibody with a fluorescent tag
Neuronal cultures-
Green – anti-MAP2
Blue – DAPI stain for DNA
Cardiomyocytes-
Blue – DAPI stain for DNA
Red – anti-troponin C
How does BMP4 affect the efficiency of glial differentiation?
Increases
How does Wnt3a affect the efficiency of neuronal differentiation?
Decreases
what is the nucleosome?
Octamer of core histone proteins: H2A, H2B, H3, H4 (2 of each)
146 bp of DNA is wrapped twice around the octamer.
what are Histone tails?
contact linker DNA and other nucleosomes
are important for chromatin folding and co-factor recruitment
how does Chromatin provide two opportunities 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 post-translational modifications that control DNA accessibility and transcription factor and RNA polymerase recruitment
what are the Post-translational modification of core histone ‘tails’?
Acetylation, phosphorylation, methylation
what is western blotting?
1 - SDS-PAGE
2 - Transfer proteins to membrane using electric current (blotting)
3 - Primary antibody
- specific to the protein being studied.
- usually raised in rabbit or mouse
4 - Secondary antibody:
- recognises the rabbit or mouse primary antibody
- amplifies the signal.
- carries the HRP (horse radish peroxidase) enzyme
what is GAPDH?
GAPDH: loading control, shows whether the same amount of protein is loaded in each lane. Do you think this gel is loaded equally? What could go wrong?
In this example, the journal article just shows a strip of the blot at the relevant size. It’s better to show the full blot. Why do you think this might be better?
what do co-factors bind to?
histone modifications for activation, folding and repression
what is gene expression regulated by?
its chromatin environment
how are HAT (histone acetyl transferase) complexes usually recruited?
by transcription factors
how are HDAC (histone deacetylase) complexes usually recruited?
by repressive transcription factors
how are Acetylated lysine residues bound?
by bromodomain-containing proteins
Some bromodomains are inhibited by drugs called BET inhibitors, currently in clinical trials for several cancers
what are epigenetic writers, readers and erasers in acetyl lysine and methyl lysine?
writer-
Acetyl lysine - HAT – histone acetyl transferase
Methyl lysine - HMT – histone methyl transferase
eraser-
Acetyl lysine - HDAC – histone deacetylase
Methyl lysine - KDM – lysine demethylase
reader-
Acetyl lysine - bromodomain
Methyl lysine - chromodomain
what is transcription regulation?
Trancription factors, chromatin structure, histone modifications, DNA methylation, long non-coding RNA, RNA polymerase factors, splicing machinery
what are epigenetic writers?
HMT and HAT
If valproic acid inhibits histone deacetylases, what will happen inside the cell when the drug is added?
More acetylation overall, so more transcription and More acetylation overall, but some genes go up and some go down
what does acetylate lysine recruit?
The acetylated lysine recruits protein complexes that have bromodomains, such as co-activators.
These promote the binding of other transcription factors and the mediator complex, leading to RNA pol II recruitment and formation of the pre-initiation complex (PIC)
look at the screen shots for lysine methylation and acetylation
what is Assaying for neuronal differentiation of stem cells?
spatial information
single cell information
average of the whole sample
easier to quantify
The DNA in one cell is about 1.5 m long.
How is it folded and organised inside the nucleus?
Metaphase chromosome, associated with sister chromatid
Interphase: Chromosome territory
Topologically associated domains
how is methyl lysine bound?
Methyl lysine is bound by chromodomain-containing proteins
what is lysine methylation associated with?
Lysine methylation can be associated with activation or repression
case study example…
My PhD student, Sylvia, wasn’t happy with the efficiency of neural stem cell to neuronal differentiation in her system. She wanted more neurons to be produced so she can study them.
After reading the literature, she hypothesised that adding a signalling molecule called Wnt3a should help produce more neurons. She also found out that the signalling molecule BMP4 should stimulate glial cell production
