Lecture 31 Flashcards Preview

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Flashcards in Lecture 31 Deck (22):
1

RNAseq:

- 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

2

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

3

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

4

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

5

Nuclear run-off:

- Similar experiment all performed in vitro

6

ChIP:

- 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

7

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

8

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

9

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)

10

Protein abundance and protein function:

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

11

Proteomic approach:

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

12

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

13

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

14

Measuring expression in the context of regulation:

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

15

Gene product function can be controlled:

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

16

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

17

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

18

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

19

A cell type:

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

20

A/aplha cell types:

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

21

alpha cell type:

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

22

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