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- Measure steady state levels of genes in the form of cDNA or RNA
- Can study gene structure
- RPKM: reads per kilobase per million reads
- You can determine what cells and what conditions produce the same reads, and can identify what genes are transcribed in what situation
- This can identify missed intron annotation, missed genes and exon branch sites depending on the condition
- Identifies gene boundaries nucleotide by nucleotide


Transcription initiation is closer to gene regulation. Measure this with RNApol2 on DNA or using ChIP;

- Measure actual rates of transcription as this is the direct output of gene regulation
- Looking for the recognition of promoters by RNAP2
- A single gene can be studied over different time periods, to see when RNAP accumulates in a paused stance


What did ChIP uncover in regard to RNAP2 binding?

- RNAP2 sits at the promoter waiting for another signal
- The TF recruits RNAP early, but it doesn't transcribe, so it is ready to transcribe at very short notice


Nuclear run-on assay (similar principle to ChIP):

- Transcription in vivo, but performed in vitro
- Lyse two cells on ice (one in the un-induced state, the other induced)
- Pellet nuclei
- Add NTPs (one radioactively labelled) and buffer to nuclei
- Incubate at 37 degrees for several minutes
- Isolate radio-labelled RNA
- The output is a reflection of the amount of transcription occurring in your gene of interest
- Only the transcripts that have already been bound by RNAP will transcribe, so the only ones we will pick up are the rounds of transcription that have already started


Nuclear run-off:

- Similar experiment all performed in vitro



- Cross link protein to DNA in living cells with formaldehyde
- Break open cells and shear DNA
- Add primary antibody of interest
- Add antibody binding beads
- Immunoprecipitate to enrich for fragments bound by protein of interest
- Reverse cross-links and treat with proteinase K (remove protein component, and left with DNA fragments bound by RNAP)
- Detect and quantify precipitate DNA through PCR and hybridisation methods


eg) One gene, A, and want to know the occupancy of RNAP at different conditions:

- Primers will amplify up the fragments
- You know that your target DNA under non-induced conditions will give you a small band (little bit of RNAP)
- Under induced conditions you will get a larger band, as there will be more RNAP bound at the promoter
- This is a measure of transcription rates, because it is looking at RNAP sitting at the promoter


ChIP can be performed on a genome wide scale:

- Do the same experiment using sequencing libraries, remove the PCR step and replace with a next generation sequence
- Make a sequencing library that you can run on a platform
- Measure the level of every fragment RNAP is bound to
- A density of map is produced with the density of reads across the genome
- Complements RNAseq, but looking at specific elements of the transcription process


Transcriptional fusion:

- Reporter genes (gfp, or GUS etc) are fused to promoters of gene of interest
- Indirect measure of transcription by measuring reporter gene product
- Qualitative cell or tissue specific expression, but not quantitation
- Which parts of the promoter are important for the expression of your gene of interest? (slowly chop down the promoter region)


Protein abundance and protein function:

- Wester blots, immunoprecipitation
- Enzyme activity and immunfluoresecence and epitope tags


Proteomic approach:

- Separate total protein content by two dimensional gel electrophoresis, identify proteins by mass spectrometry
- This is two dimensional separation of proteins


Protein interaction studies:

- Proteins usually operate in complexes
- The gene of interest is translationally fused to TAP-tag, purify and then detect
- Calmodulin beads pull out the interacting factors as a pure protein complex
- Mass spec will determine exactly what proteins are acting
- Temporal and spatial investigation


Gene expression in whole organisms:

- Tagging proteins in the embryo can allow us to understand proteins involved in cell development over time
- Spatial changes in gene expression related to localisation and specification


Measuring expression in the context of regulation:

- DNA to RNA to the cytoplasm where proteins are expressed that perform a specific action.


Gene product function can be controlled:

- Through activation based on location (cytoplasm vs nucleus)
- Processing, degradation, targeting etc


Protein modification:

- Phosphorylation (kinase) and de-phosphorylation (phosphatase)
- A hydroxyl group gets phosphorylated very specifically which usually results in activation (sometimes this inactivates it)
- De-phosphorylating results in the reverse action
- Common regulation in the cell cycle
- Many types of kinases - this is a major method of regulation in eukaryotes


MAP kinase:

- A way for the cell to sense the external environment through a sensor in the cell membrane and transmit this signal through the MAP kinase cascade to promote a gene response
- A change of phosphorylation results in the genetic action
- Pheromone response in yeast
- Growth factors, cytokinase and cell stress pathways in mammals


Saccharomyces cerevisiae vegetative growth cycle to do with gene expression:

- The mother cell buds to give daughter cells
- Two mating types, a or alpha, and the two cells can recognise each other
- In response to pheromones (which attract the opposite type of mating type), which causes the actin/microtubule to move toward the source of the pheromone
- Schmoo: the actin cytoskeleton of different cells move toward the source of the pheromone, as part of the actin cascade
- Cell fusion produces one big cell of a and alpha, which becomes a diploid cell, which can grow vegetatively to produce more a/alpha cells
- Under nutrient limitation sporulation follows (an ascus forms filled with both cell types as ascospores) and they grow vegetatively


A cell type:

- Expressed a specific genes, but not alpha specific genes
- Expresses mating genes and pheromone production genes


A/aplha cell types:

- Only expression the sporulation and meiosis genes, none of the other genes!


alpha cell type:

- Expresses alpha specific cells, with mating genes an pheromone production which are specific for alpha cells


Mating signalling cascade:

- Operates via a MAPK cascade
- A set of genes encoding pheromones (cell specific) and all of the MAPK genes, and a TF
- Epistasis experiments helped produce a model for signal transduction
- Pheromone binds receptor and conformational change occurs, P21 activated kinase is phosphorylated
- A TF is phosphorylated, which initiates gene expression of genes involved in mating
- Genes that block mitotic division is G1 are expressed, as are FUS1 cell fusion genes are expressed