RR15: System Biology Approaches

Question 1

What is the basic cellular toolkit?

Answer 1

It’s the common material that’s present in each organism and it’s highly conserved.

Question 2

Do we know the function of all the genes?

Answer 2

No. 50% of the genes are of unknown function.

Question 3

How can we figure out what’s the function of a gene in an organism?

Answer 3

By disrupting 1 gene product on 1 chromosome of a diploid yeast.
When we disrupt or take out a gene, by comparing the phenotype of the wild and the mutant types, we can understand the role of a gene.

Question 4

How can we modify the genome of a yeast?

Answer 4

1. Make sure the yeast is diploid
2. Using homologous recombination, transform one gene on one chromosome with a disruption construct
3. Select your disruption construct for G-418 resistant, so it can grow on drugs.
4. The haploid progeny will have the mutant or wild type chromosome.
5. We can assess the effects of the gene replacement by the viability or the growth rate.

Question 5

How can we make homologous recombination?

Answer 5

Yeast will recombine very quickly homologous sequences.
1. Know the sequence information on the ends of the gene we’re interested in
2. Make a construct that has 100% homologous sequences on the ends.
3. Use a PCR reaction with primers that correspond to those homologous regions.
4. Amplification of a dominant selectable marker, like an antibiotic drug-resistant.
5. You end up with a drug-resistant segment that has homologous sequences to the gene of interest.
6. Introduce this segment in the cell to have a homologous recombination.
7. Present the drug to the cell, so all the cells that didn’t take the disruption construct will die, so we can focus on what we’re interested in.

Question 6

After the homologous recombination of the gene of interest with the disruption construct, what do we do?

Answer 6

We can present the drug to the cells, so only the ones with the disruption construct can live.
Then, we make the yeast sporulate to have gametes that will be haploid with either the mutant chromosome or the wild chromosome.

Question 7

What kind of effects can you observe on mutant haploid yeast after homologous recombination?

Answer 7

If they don’t make gametes, it’s an important gene, so it’s in the basic cellular toolkit.
If they live.
If they grow slower.
Observe if they change under certain conditions.
Challenge them with sugar.
Lack of amino acids.
High temperature.

Question 8

What are flanking sequences?

Answer 8

The sequences next to the DNA sequence of interest.
Used as reference points for making primers or for homologous recombination.

Question 9

If a gene is very essential, will the diploid yeast be able to make spores when the gene is disrupted?

Answer 9

It could or it could not.
If it doesn’t make spores, it for sure tells you it’s essential, but if it makes spores, it can still be essential, but we can find this out by doing a bunch of tests under various conditions.

Question 10

How can we find out what a gene does in a functional genome, not just in one cell?

Answer 10

We can use RNAi.
It can affect all kinds of different cells in real-growing organisms and we don’t have to wait to see how the progeny will look like, like with specific homologous recombination.

Question 11

Why is RNAi so efficient in C. elegans?

Answer 11

First, because C. elegans can eat bacteria that have the dsRNA we want to introduce in the organism.
Also, when RNA gets into C. elegans, it goes through an amplification process, so RNA because more abundant and it goes in all tissues (except neurons)

Question 12

How can investigators analyze every single gene function in C. elegans?

Answer 12

1. Engineered plasmids that would each drive a dsRNA.
2. A promoter would drive the expression of one RNA strand one way, and another promoter would drive the expression of the other strand the other way.
3. You make 19 000 different constructs and they will all make a single predicted gene in C. elegans.
4. You feed those constructs to bacteria
5. You feed those bacteria that carry the plasmid expression dsRNA to C. elegans. (one each)
6. You observe every organism while they show the effect of loss of function of that particular gene.

It allows us to know what the function of the genes are, even the gene functions that were unkown.

Question 13

By looking at the gene functions of the entire genome of C. elegans, what can be useful for humans?

Answer 13

We can see phenotypes of different types, like an animal that lost coordination of movement.
That gene is associated with the neuromuscular function which are highly conserved in all organisms.
So we can understand, that this specific gene in C. elegans that is affecting movement coordination would do a similar function in humans.

Question 14

How can we use proteins to discover the function of the genes?

Answer 14

Transcription factors are modular, they have DNA-binding domains and transcriptional activating domains.
1. Fusing a protein of interest to a DNA binding domain for which you know the DNA binding sequences (like GAL4 binds to UAS)
2. Fusing a different protein that you think might interact with the first one to a transcriptional activator.
3. So, if protein 1 and 2 interact, in fact, with each other, it will activate the transcription of any gene that has the seqeunce corresponding to the DNA binding domain (UAS)

If they interact, it grows, if they don’t, it doesn’t grow.

Question 15

What do we mean when we say that transcription factors are modular?

Answer 15

It means that transcription factors have the ability to bind to DNA via their DNA binding domains while also binding to other transcriptional regulatory proteins.

Question 16

What’s the 2 hybrid system?

Answer 16

It’s a way to identify protein-protein interaction by fusing one protein on a DNA-binding domain and the other on a transcriptional activator.
If they interact, we will have the expression of the downstream gene.

Question 16

When we use the fusion of proteins on the DNA-binding domain and on the transcriptional activator, what do we call these 2 factors?

Answer 16

DNA-binding domain + protein of interest: bait
transcriptional activator + protein we think interacts with the protein of interest: prey

Question 17

You can also test where the proteins come together using the fact that transcription factors are modular. How?

Answer 17

By knowing the 2 proteins on the DNA-binding domain and on the transcriptional activator, we can evaluate which cells grow and figure out what the gene product attached to the transcriptional activator is.

Question 18

What’s the 2 hybrid system based on protein fragment complementation?

Answer 18

It’s when the bait and the prey come together, but they each have one half of a protein. One has the C-terminal, the other has a N-terminal.
When the bait and prey come together, the protein comes back together and it might have an actual function.

Question 19

What’s one protein that can be used during 2 hybrid system based on protein fragment complementation?

Answer 19

Dihydrofolate reductase.
It’s really important for purine synthesis, without it you can’t grow.
So if the bait and prey come together, they will reconstitute dihydrofolate reductase and the cell will be able to grow again.

Question 20

How does 2 hybrid system based on protein fragment complementation work with GFP?

Answer 20

GFP is split in 2 parts. The 2 parts by themselves don’t give rise to fluorescence.
But if the 2 parts are coming together with the bait and prey, we will have fluorescence by reconstituting the GFP protein.
With that, we can detect which proteins are interacting because when they do, we see fluorescence.

Question 21

How can we know gene function by 2 hybrid system?

Answer 21

By knowing which proteins interact with each other, it can help us understand what the functions of their given genes are. Usually, proteins that are working together have similar functions.

Question 22

What are the conditions for yeast 2 hybrid analysis?

Answer 22

The proteins have to go in the nucleus where they’re going to function. But the problem is some proteins don’t like the nucleus, but that’s why we use protein fragment complementation, so the protein doesn’t have to be whole before entering the nucleus.

Question 23

What’s the difference between 2 hybrid analysis and 2 hybrid based on protein fragment complementation?

Answer 23

2 hybrid analysis is about finding what 2 proteins can interact together. When they do interact together, the DNA-binding domain and the transcriptional activator will be able to activate the transcription of the DNA.
The protein fragment complementation is that instead of having 2 proteins interact with each other, we bring 2 halves of the same protein, so the protein can then do its function.

Question 24

What’s proximity labelling?

Answer 24

It doesn’t require protein-protein interaction.
You put a specific activity on the protein of interest that will put a label on all the protein that come close to it.

Question 25

How can you make a protein that will be able to do proximity labelling?

Answer 25

Make a fusion protein with biotin ligase.
Biotin: small vitamin B
Mutant biotin ligase can add a biotin to any protein with a primary amine.
By putting the protein of interest and the biotin ligase in a plasmid, and making it interact with biotin, we can have a construct that will express the protein in the cell. Since the protein is now fused with biotin ligase, it will add a biotin to any protein that comes close to it.
The proteins that come close to the protein of interest have a biotin tag now.

Question 26

After doing a proximity labelling, how can we isolate those proteins that have a biotin tag on?

Answer 26

By using affinity chromatography with streptavidin that has a high affinity with biotin, the biotin on the protein will bind to the streptavidin molecules.
Wash the column to get rid of everything we don’t want.
Elute all the biotinylated proteins by adding biotin.
Biotin will create competition for the streptavidin molecules, so the biotinylated proteins will fall off.
Then, you have proteins that went close to the protein of interest.
With mass spectrometry, we can get the identities of those proteins.

Question 27

What does proximity labelling tell us?

Answer 27

It tells us which proteins are in close proximity to our protein of interest which can give us a good idea of what their function might be.

Question 28

How can we get a network of proteins that interact directly and indirectly with our protein of interest?

Answer 28

By doing 2 hybrid analysis, we can figure out which 2 proteins are interacting directly with each other and with proximity labelling we can tell which proteins are in close proximity with each other. Knowing which proteins are close to the protein of interest can tell us that 2 of those proteins might be interacting with eachother, and it gives rise to a network of proteins.