RR15: Systems Biology Approaches Flashcards
1
Q
what is very useful property of yeast?
A
yeast cells very quickly recombine sequences that are homologous
2
Q
how can the yeast genome be modified to test for the function of a gene?
A
- yeast lends itself to genetic analysis
- First, you have to make the yeast diploid so that you have chromosomes because you’re going to disrupt a gene product on the other chromosome
- You can do that by virtue of yeast cells very quickly recombining sequences that are homologous (property that is very useful)
- if you have a gene you’re interested in, you only need is a bit of sequence information on the flanking end of those genes (20-40 nucleotides)
- Make a construct that consists of that flanking sequence that is 100% homologous to the gene you want to eliminate or disrupt, you can carry out a PCR reaction with primers that correspond to those flanking homologous regions and that will allow you to amplify the sequence of a dominant selectable marker: drug/antibiotic resistance gene
- if the cells have one copy of that resistance gene that can grow: dominant selectable marker
- the drug resistance gene will eventually have the homologous ends that look exactly like the gene that is want to eliminate in the flanking regions
- can be very easily introduced into the yeast cell because they are easy to transform/transfect
- Once that disruption construct/fragment is in the cells, it will cause a recombination event between those flanking sequences thereby eliminating the target gene and replacing it with teh ddominant selectable marker, all by homologous recombination
- if you grow the cells in the presence of the drug, the cells that didn’t carry out this process will die
- That leaves you with yeast cells that have one normal chromosome and one engineered chromosome (where the gene is disrupted), could be any gene that is unknown
- Get the yeast to sporulante: make its gametes, become haploid again
- Examine what it means for this cells when they lack that function
- if no gametes are made, it is part of the basic cellular toolkit
- if they do make gametes you can grow them and test them in all kinds of conditions to see what that gene does exactly
- it will tell you something about teh contribution of that gene in the various contexts
- why you start with a diploid: you need a backup chromosome there that is not affected in case the gene you eliminated is essential and the cell dies
- you can only see that when it sporulates
3
Q
how can a genome wide analysis of C.elegans be done?
A
- all you have to do is get the dsRNA in to remove the gene that you want
- you could analyse all the unknown genes
- Even better in C.elegans is that the RNA introduced will fo through an amplification step, so you now have a lot, and that is systemic
- It gets into all tissues except neurons
- Come up with a genome wide way of analyzing all the genes in C.elegans (19,000)
- Engineer plasmids so that each plasmid would drive a dsRNA, first by having a T7 promoter that drives the expression of one strand of RNA in one direction and another T7 promoter driving RNA expression in the other direction
- T7 RNA polymerases are not normally present in bacteria but you can make them present and induce their expression with IPTG
- IPTG is a way of activating the T7 promoters that will make the dsRNA
- Make 19,000 different constructs, each of which makes dsRNA that corresponds to a single predicted gene in C/elegans
- Transform 19,000 independent colonies, and feed each one of them to the worms
- Each will show an RNAi effect typical of loss of function of that one gene, so you can figure out what every gene does
4
Q
what are the genes that go into the basic cellular toolkit class?
A
- Genes that give rise to sterile animals or cause embryonic lethality are involved in the class of genes that are part of the basic cellular toolkit (DNA synthesis, RNA metabolism, translation, transcription)
5
Q
what genes are involved in neuromuscular function?
A
- things that made the animal not move properly, made the animal uncoordinated: genes involved in neuromuscular function (many of which are conserved up to humans)
6
Q
what genes control where and when organs are made?
A
- post embryonic phenotypes are involved in signaling so that organs are formed in the correct time and place, these are more animal specific and not necessarily part of the basic cellular toolki
7
Q
what is a more proteomic approach that can be taken?
A
- take advantage of the fact that transcription factors are modular: have different domains (binding and activation)
- As long as they are put together, doesn’t matter what factor they come from
- They will always activate the downstream gene based on the DNA sequence that it interacts with
8
Q
what is a way to make fusion proteins to test for protein:protein interaction?
A
- can make fusion proteins: protein of interest A fused to a DNA binding domain that is very well characterized, and you know the DNA sequence that it binds to (like GAL4 binding to UAS)
- if you think that it might be interacting with protein B, make a fusion with the transcriptional activation domain that is well described
- if you put them together, they will activate transcription
- If A and B interact together, they’ll join the two domains together and activate the transcription of that gene
- Way of selecting for a clear protein protein interaction
- If no interaction, cells dont grow (in the case of histadine)
- Test it with different reporter genes
9
Q
what is the bait?
A
- Bait: protein that you’re interested in bound to a DNA binding domain
10
Q
what is the prey?
A
- prey: protein that you’re going to query that’s bound or fused to a transcriptional activation function
11
Q
what are the limitations of the two hybrid system?
A
you have to put it in the nucleus, its hard to look at all those proteins at the same time etc
12
Q
how does protein fragment complementation work?
A
- Bait and prey come together and reconstitue a protein that has been separated into two parts (C and N terminal)
- If they come together, they will reconstitue a protein and it will have an actual function
- using a protein called dihydropholate reductase
- If protein X and protein Y come together, cells can grow again
- You even do it with GFP so that GFP is engineered so that it is split in two: the two parts themselves dont give rise to fluorescence but if they are brought together they will constitute a proper GFP protein
- All of that gives you an idea of which proteins interact with which proteins
13
Q
what do proteins that interact together have?
A
they have similar functions = can help tell us about the function of an unknown protein
14
Q
what is Bio-ID used for?
A
to identify what proteins your target protein interacts with
15
Q
how do you do Bio-ID?
A
- Proximity labeling: doesn’t necessarily require a direct protein protein interaction
- Special activity put on protein of interest that will label all the proteins that come close to it during a period of time
- Make fusion proteins with protein of interest (bait) and an enzyme clalled a biotin ligase
- Biotin are small molecules that are important for normal cellular processes and have to be ligated to specific proteins on primary amines by biotin ligament
- usually very specific and only have a few targets
- By mutagenesis, we can create a very promiscuous biotin ligase that no longer has the 4 or 5 substrates, but it will biotinylate any protein with a primary amine
- If you take expression contract that will express this fusion protein and have it express, the promiscuous ligase will biotinylate any protein that comes to a close range to the protein that you’re interested in
- The proteins in the range will have a biotin tag
