Part 3: Gene regulation

Question 1

What makes cells different?

Answer 1

Every cell type has proteins specific to it, such as haemoglobin in RBC

Question 2

How is differential gene regulation achieved during development?

Answer 2

1) Transcriptional regulation

2) Post-transcriptional regulation of protein synthesis and function

Question 3

How do we know that transcriptional regulation is the major mechanism for differential gene expression?

Answer 3

Only certain types of mRNA are found in specific tissues, very few mRNA are present in all cells

Question 4

RNA Northern blot

Answer 4

1) Isolate RNA from tissue
2) Separate by size using electrophoresis
3) Transfer to hybridization membrane
4) Hybridize labelled probe to the membrane containing RNA

Question 5

Advantages and disadvantages of Northern blot

Answer 5

+ Tells size and abundance of the transcript

- Time-consuming

Question 6

Reverse transcriptase (RT-) PCR

Answer 6

1) Isolate mRNA from tissue
2) Make cDNA using reverse transcriptase
3) Run PCR using gene-specific primers
4) Check the presence of the gene of interest on the gel

Question 7

After a RT-PCR reaction, you run your PCR product on an agarose gel. What determines the size of the DNA amplified by RT-PCR?

a) The size of the transcript
b) The primers used in RT-PCR

Answer 7

The primers used in RT-PCR will determine the size of the amplified DNA fragment since they tell polymerase where to start and to stop

Question 8

Advantages and disadvantages of RT-PCR

Answer 8

+ Fast and sensitive

- No information about size, subject to artifacts, crudely quantitative

Question 9

How to get a quantitative result from PCR?

Answer 9

Use Real-time PCR and detect the difference of DNA produced during exponential phase

Question 10

In situ hybridization

Answer 10

1) Isolate tissue
2) Hybridize gene-specific probe to the tissue on the slide
3) If transcripts of gene of interest are present in some of the cells, probe will be hybridized and detected

Question 11

Advantages and disadvantages of In situ hybridization

Answer 11

+ Gives information about transcript spatial distribution

- Does not tell the amount, time-consuming and difficult technique

Question 12

You are going to compare the expression levels of an Arabidopsis microRNA (21 nucleotides) between root and leaf tissues. Which one of the following methods is best suited for your experiment?

a) RNA blot
b) Quantitative RT-PCR
c) In situ hybridization

Answer 12

RNA blot
RT-PCR won’t work due to small transcript size
In situ hybridization gives no information about the amount

Question 13

Why traditional RT-PCR is not very quantitative?

Answer 13

After several cycles, reaction reaches plateau which is not correlated with the amount of DNA present (reagents run out)

Question 14

Microarray hybridization

Answer 14

1) Synthesize large amount of probes for different genes
2) Array the probes on the hybridization membrane or array
3) Isolate RNA from tissue
4) Make labelled cDNA
5) Hybridize cDNA to probes and detect signal

Question 15

Advantages and disadvantages of Microarray hybridization

Answer 15

+ Provides information on the amount of every gene detected

- Expensive, result must be verified by another technique

Question 16

RNA sequencing

Answer 16

1) Isolate RNA from the tissue
2) Make cDNA using reverse transcriptase
3) Sequence

Question 17

How does sequencing work?

Answer 17

1) Fragment DNA
2) Ligate adaptors
3) Array of PCR using Bridge PCR
or
3) Enzymatic extension with fluorescently labelled nucleotides

Question 18

Advantages and disadvantages of RNA sequencing

Answer 18

+ Sensitive, quantitative accurate, provides information on all transcripts
- Expensive

Question 19

Promoter-reporter gene fusion

Answer 19

1) Construct chimeric gene using reporter gene (tagged) and a promoter and regulatory sequence of the gene you want to study
2) Transform into the organism in a way so that all cells carry this chimeric gene
3) See which cells express the reporter gene

Question 20

Restriction enzymes: 3 features and 2 uses

Answer 20

Cuts DNA at restriction sites
These sites are often palindromic
Can leave either blunt or sticky ends
Fragment DNA to small pieces to make it easier to study
Create constructs by religating different DNA sequences

Question 21

How are chimeric genes introduced into the organisms?

Answer 21

Using cloning vectors, such as plasmids, that are able to replicate within an organism

Question 22

Advantages and disadvantages of Promoter-reporter gene fusion

Answer 22

+ Shows where the gene of interest is transcribed

- Time-consuming, need to know regulatory sequence

Question 23

Describe the structural and functional difference of transcriptional and translational reporter

Answer 23

Structural: transcriptional construct only has a regulatory sequence and label sequence, such as GFP, whereas in translational there is also a coding region of the protein of interest.
Functional: transcriptional reporter is used to see where the gene is transcribed, translational is used to map the location of the protein in the tissue

Question 24

Which techniques are used for global and for individual gene analysis

Answer 24

Global: Microarray and RNA sequencing
Local: RNA blot, qPCR/RT-PCR, In situ, promoter-reporter gene fusion

Question 25

What is the principle mechanism of transcript level control?

Answer 25

Regulation of transcription through enhancer and promoter regions (regulatory sequence controls gene X, but when substituted with Y the localization pattern remains the same as for X)

Question 26

Transcription factors facts

Answer 26

Influences RNA Polymerase to decrease or increase transcription 10-20% of all genes code for TF Mutations during development are often due to the TF mutations or other regulatory proteins

Question 27

Define anterior and posterior

Answer 27

Anterior is what is at the front, posterior is what comes after

Question 28

How TFs regulate the transcription?

Answer 28

They bind, either alone or as a complex, to the cis-acting enhancer elements of the target gene's promoter region. In cells lacking TF no transcription of the target gene takes place. TFs can form different complexes and, depending on the combination, will regulate transcription of different genes. TFs are controlled by other TFs (time) AND environment (time and place)

Question 29

What kinds of evidence do you need to prove that | gene Y is a target gene of transcription factor X?

Answer 29

1. Mutating X affects the expression of Y 2. X can bind to cis elements in the regulatory sequence of Y in vitro 3. AND in vivo (Chromatin immunoprecipitation-PCR, ChIP-PCR)

Question 30

How does Next Generation Sequencing differ from older techniques?

Answer 30

It is not based on the electrophoresis

Question 31

How does LacZ work in detecting location of the protein as a reporter gene?

Answer 31

It codes for b-galactosidase which converts clear substrate to blue --> visual detection

Question 32

You suspect that transcript of a newly cloned C. elegans gene is located specifically in the developing nervous system. Which of the following techniques would be the best choice to confirm your hypothesis? a) RNA blot b) RT-PCR c) In situ hybridization d) Microarray hybridization e) Promoter-reporter gene fusion transgenic C. elegans

Answer 32

Promoter-reporter or In situ

Question 33

How are TFs controlled?

Answer 33

1) Transcriptionally 2) Post transcriptionally - alternative splicing - RNA stability and localization - translation inhibition 3) Post translationally - Phosphorylation - Degradation - Proteolysis - Allosteric regulation - Protein localization