Lecture 31

Question 1

RNAseq:

Answer 1

• 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

Question 2

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

Answer 2

• 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

Question 3

What did ChIP uncover in regard to RNAP2 binding?

Answer 3

• 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

Question 4

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

Answer 4

• 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

Question 5

Nuclear run-off:

Answer 5

• Similar experiment all performed in vitro

Question 6

ChIP:

Answer 6

• 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

Question 7

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

Answer 7

• 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

Question 8

ChIP can be performed on a genome wide scale:

Answer 8

• 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

Question 9

Transcriptional fusion:

Answer 9

• 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)

Question 10

Protein abundance and protein function:

Answer 10

• Wester blots, immunoprecipitation

- Enzyme activity and immunfluoresecence and epitope tags

Question 11

Proteomic approach:

Answer 11

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

Question 12

Protein interaction studies:

Answer 12

• 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

Question 13

Gene expression in whole organisms:

Answer 13

• 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

Question 14

Measuring expression in the context of regulation:

Answer 14

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

Question 15

Gene product function can be controlled:

Answer 15

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

Question 16

Protein modification:

Answer 16

• 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

Question 17

MAP kinase:

Answer 17

• 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

Question 18

Saccharomyces cerevisiae vegetative growth cycle to do with gene expression:

Answer 18

• 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

Question 19

A cell type:

Answer 19

• Expressed a specific genes, but not alpha specific genes

- Expresses mating genes and pheromone production genes

Question 20

A/aplha cell types:

Answer 20

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

Question 21

alpha cell type:

Answer 21

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

Question 22

Mating signalling cascade:

Answer 22

• 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