LECTURE 9 (Analysis of gene expression)

Question 1

What do we need to control in gene expression?

Answer 1

• transcriptional control
• RNA synthesis control
• RNA transport control (from nucleus to cytoplasm nothing has been damaged)
• translation control
• protein activity control

Question 2

How do we measure the rate of transcription?

Answer 2

Technique called run off transcription.

1. isolate the nuclei from cell you want to study
2. once this is isolated, transcription is frozen however you can add dNTPs (and radiolalbelled UTP), that will incorporate and transcription continues The key here is to understand that once to have isolated the nuclei, there is no “re-initiation” of transcription. So the RNA polymerase complexes will “simply” “run-off” the transcripts (i.e. finish what they had started transcribing) if you provide the necessary ribonucleotides for them to do so.
3. because you used radiolabelled UTP, this tells you how many RNAP were needed The more active the transcription the greater the number of RNA polymerase complexes on the gene.
4. finally you put the DNA in a membrane, hybridize with mRNA created and get radioactivity and by measuring the radioactivity you get an idea of how transcriptionally active the gene was. the products of the reaction are hybridized to target DNA to estimate the amount of specific genes.

Question 3

Why is it called run-off?

Answer 3

because it’s done in vitro and transcription was switched off and RNAP will finish what they were doing by adding the correct dNTPS.
It’s run-on when it’s done in vivo.

Question 4

Apart of run-off method, can we measure the rate of transcription in a different way?

Answer 4

yes, we can use reporter genes, this is based on the assumption that the rate of transcription is determined by the promoter seq. isolate the promoter and fuse it with a reporter gene, introduce that into a cell and treat it with different concentrations and then measure the activity of the reporter gene that will be directly related to the rate of transcription

Question 5

What is the assumption taken in order to use reporter genes for measuring the rate of transcription?

Answer 5

That promoter seq determine the rate of transcription

Question 6

How do we measure the transcriptional abundance i.e. the amount of RNA produced ?

Answer 6

• qPCR
• In situ hybridization
• RNA protection assays.

Question 7

Describe in situ hybridization.

Answer 7

you slice the tissues that you want to analyze and use labelled antisense (fluorescently or radiolabelled) probes that are complementary to the gene of interest. you can see where RNA is more concentrated in an image.

Question 8

Why can we use RNA protection assays to measure the abundance of RNA?

Answer 8

because we can carry them and the intensity of the band will show how much RNA there is

Question 9

Why can we use qPCR to measure the abundance of RNA?

Answer 9

The more product we get with a sample the higher the intensity therefore the more initial RNA qPCR can give a measure of the absolute quantity of the target polynucleotide

Question 10

What is FRET?

Answer 10

Fluorescent resonance energy transfer. you have two oligos that are close enough and its energy can be transferred if the one emitting green light is close enough to the red light one then the fluorescence emitted is red.

Question 11

Why is FRET useful in qPCR?

Answer 11

because it can be used to reduce the rubbish produced because you create probes that are complementary to an internal sequence and this probe consists in two oligo a with two different fluorescent proteins

Question 12

What is trascriptomics?

Answer 12

Transcriptomics is the study of the transcriptome—the complete set of RNA transcripts that are produced by the genome, under specific circumstances or in a specific cell—using high-throughput methods, such as microarray analysis. Comparison of transcriptomes allows the identification of genes that are differentially expressed in distinct cell populations, or in response to different treatments.
a discipline that makes use of gene chips to study gene expression
the large-scale study of RNA abundance

Question 13

What does it mean the term “gene expression”? (3)

Answer 13

A process that includes transcription, RNA processing and translation

A process that can include modification of a protein after translation

The process whereby a gene is activated to, ultimately, determine the phenotype of the gene itself

Question 14

How do we measure global level of gene expression?

Answer 14

we use microarrays, big datasets. you measure fluorescence to see how abundant genes are
however massive datasets are difficult to analyze

Question 15

What is spectroFLORIMETRY?

Answer 15

a technique to measure the quantity of label bound to the probes on the array

Question 16

describe microarrays.

Answer 16

Microarrays: a plate with chips made by robots, in each chip there’s a DNA seq and the you add probes (RNA) that should hybridize with the homologous DNA. When this happens fluorescent spot appears. the different levels of hybridization are determined by different colors. main aim is to ,easure the expression of large datasets of genes however it can be used for quantifying duplications or deletion or for detecting protein-DNA interactions

Question 17

What type of microarrays can we find?

Answer 17

cDNA and oligonucleotides microarrays.