LECTURE 4
Learning points.
1. How does one do a high copy screen and what types of things do you get out.
2. Starting with a mutant or gene in hand, you should understand how you would go
about performing a second site suppressor screen and a synthetic lethal screen.
3. You should understand the different ways one mutation might suppress the phenotype
of another mutation. You should also understand how you might distinguish among
these.
4. You should understand why two mutations could generate a synthetic lethal phenotype.
References
A large scale synthetic lethal screen.
Tong AH, ………..and Boone C. Global mapping of the yeast genetic interaction
network.
Science. 2004 Feb 6;303(5659):808-13.
Pan… Boeke (2004)“A robust toolkit for functional profiling of the yeast genome.” Mol.
Cell 16 p487-496
3) synthetic lethality. Lets say that we have a t.s. mutant that is unable to bud. It
is a kinase with 10% activity at 23°C (which is enough to get by), and 1% activity at
36°C (not enough). Image that this kinase has another subunit, or there is another
member of this pathway upstream or downstream. If we also have a hypomorphic
(weakened, but not dead) mutation in this other member of the pathway, we may drop the
overall activity of this pathway to a level where the cells are just dead at any temperature.
-this is called synthetic lethality. (note synthesis meaning you are creating something by
bringing two things together).
First, how would this look in a cross:
MATa yfg1-1 YFG2
x
(no linkage)
MATa YFG1 yfg2-1
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or:
aB x
Ab
THERE ARE MANY POSSIBLE REASONS for SYNTHETIC REALITY:
(balls are individual proteins)
Mutations can be within a single linear pathway, or two parallel pathways.
There might be two or more copies of a single gene.
-CLN1 and CLN2 are very similar to each other, and somewhat similar to CLN3.
You can delete any two, but not all three.
Loss of a gene’s function could alter the physiology of a cells in such a way that the cell
might require a pathway that is not normally essential.
-Hypomorphic alleles of many replication proteins (ts alleles at 23°C) are
synthetically lethal with mutations in the DNA damage checkpoint pathway. That is
because this is the pathway that responds to DNA damage that occurs with high
frequency in the replication mutants.
Synthetic lethality can be examined with non-essential genes or with hypomorphic
alleles of essential genes. Almost all ts alleles are hypomorphic at the permissive
temperature!!!!!!
How many of the possible synthetic lethal scenarios shown above can be ruled out if
a deletion is used? Very few. Of the above, one can rule out a situation such as that
between mut1 and mut2.
-It is surprising (at least to me) how often essential complexes, containing proteins
encoded by many essential genes, will have non-essential subunits.
One can extrapolate everything that we did to cover synthetic non-lethal
phenotypes: sterility, sensitivity to a drug, etc.
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Systematic synthetic lethal screens:
the basic idea
yfg1∆yfg2∆ ?? yfg2∆?
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How can this be ameliorated?
Integrate MFA1pr-HIS3 at the CAN1 locus.
-> can1∆::MFApr-HIS3
CAN1 encodes the arginine permease.
Canavanine is an arginine analog that kills cells.
Therefore, can1∆ cells are resistant.
IMPORTANTLY, CAN1 is shows dominant sensitivity.
Why does this help?????
Haploid selection is now –histidine +Canavanine +NAT +G418
-histidine selects for the haploid, +canavanine selects against the diploid.
WHAT IS THE USE OF SYNTHETIC LETHALITY?? WHAT DOES IT TELLS
US?
Not too much, by itself.
- Genes could be in the same pathway under some circumstances (where the mutation causes a
partial loss of function in the pathway).
e.g. cdc28-1N is synthetic lethal with deletion of one normally non-essential cyclin gene, clb2
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- Genes could be in parallel pathways that perform similar functions.
e.g. can1∆ is synthetic lethal with arg4. WHY?
- Genes could be in parallel pathways that don’t have directly related functions, but where one
is required when the other is missing.
e.g. mutants that disrupt replication cause DNA damage. Thus, many of these mutants are
lethal with mutants in pathways that fix DNA damage. E.g. the okazaki processing
enzyme mutant fen1∆ is lethal with the DNA damage checkpoint mutant mrc1∆ and the
recombination mutant rad52∆.
THEREFORE, THIS SORT OF ANALYSIS COULD ALLOW YOU TO MAKE
EDUCATED GUESSES AT THE FUNCTION OF A GENE.
However, what if you found a gene of
unknown function that a gene
you were interested in had the
very same set of synthetic lethal
interactions as genes in a well
studied pathway it would
strongly suggest that your gene
functioned in that pathway too.
Red boxes are synthetic
lethalities.
Because of the way this is
done, it is sometimes hard to
distinguish synthetic very
sick, from lethal.
Does this matter???
Cluster IX below contains two
genes known to be required for
the replication checkpoint:
TOF1 and MRC1.
It also contained another gene
CSM3, whose function was not
known.
This led the authors to test
whether csm3∆ mutants were
also defective in the replication
checkpoint-> they are!!
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The deletion collection is also bar coded.
This means that each deletion has a unique 20 nucleotide identifier (actually, each has two).
Therefore, one can do a PCR reaction on DNA purified from any deleted strain and identify
the deletion.
The PCR primers flanking the barcode are
the same for all strains.
W
hat is the advantage to this???????
Y
ou can mix the strains and examine
changes in the ratios of the
deletions.
T
his has been used extensively for
pharmacogenomics.
hat will happen if you do this
experiment with a drug that
causes DNA damage??
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W
What would happen if you did
this experiment with FOA?
This can also be used to examine synthetic lethality.
How? You do a transformation of a set of pooled deletions with a KO construct.
You Could imagine doing it as follows:
What is wrong with this. There are two issues
D Slam. Diploid Synthetic lethal analysis on microarray.
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