YG 2015 P3
Project 3: Sorting of mutants into complementation groups
Yeast genetics is a powerful tool to identify mutations in pathways you are
interested. Depending on the complexity of the pathway you chose to investigate, and the
specificity of the mutant selection or screen you designed, you may obtain mutations in
more than only one gene. If you identified more than one mutant candidate during your
work (and you SHOULD if you worked hard enough and had a well-designed screen or
selection) you now have the problem that you KNOW that several of these candidates
probably have mutations in the same gene, but you do not know which of these. Cloning
all of these mutants by complementation with a yeast genomic library is a lot of work and
would be very frustrating if you would be fishing out the same gene numerous times.
Luckily, our yeast comes to the rescue again to save you from this frustration.
If you were smart, you carried out your screen in two isogenic strains of different
mating type. If you have a collection of mutants in both mating types, and confirmed that
the mutations are recessive, you can now identify mutation in the same gene by mating
mutants of different mating types with each other and test if the mutants complement
each other, i.e. if the mated diploid still displays a mutant phenotype (=mutants in the
same complementation group) or if it now behaves like wild type (=mutants in different
complementation groups).
We have a collection of respiratory deficient mutants. All of them are unable to
synthesize lipoic acid. This compound is a fatty acid derivative used by several
mitochondrial enzyme complexes (pyruvate dehydrogenase, alpha-ketoglutarate
dehydrogenase and glycine cleavage system). The Hiltunen group has been busy
identifying factors involved in the generation of lipoic acid. Mostly we have been
interested in the synthesis of mitochondrial fatty acids, which are required for respiration,
among other reasons because this pathway provides the octanoic acid precursor for lipoic
acid biosynthesis. The mitochondrial fatty acid synthesis pathway is conserved in
humans. We have evidence that mitochondrial fatty acids have other role(s) in respiratory
competence in addition to lipoic acid synthesis, and we have spent a good part of the last
decade on determining the function of this pathway. We will use the mutant collection for
this exercise so you can get an impression how the sorting of mutants into
complementation groups works in real life, instead of just using a printed chart.
Day 1 (Tuesday 1st week): Mating of the mutants
We have six mutants (numbered 1-6) of mating type a and three mutants (A-C) of
mating type α. All mutants carry recessive mutations causing respiratory deficiency, and
mutants 1-6 are unable to synthesize methionine (Met-) while mutants A-C cannot make
lysine (Lys -). We can use these marker mutations to select for the diploids.
Mate mutants 1-6 with mutants A-C as already described for project 2 (on YPD). Every
group does the matings in duplicate (2 x 18 matings total; six matings per plate). Leave in
30oC incubator overnight.
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YG 2015 P3
Day 2 (Wednesday 1st week) in the morning: Selection of Diploids
As mentioned before, the mutants have different markers (methionine and lysine
deficiency), allowing us to select the diploids by selecting for cells that can grow on
media lacking both amino acids. Streak out mated cells and controls for single colonies as
for project 2, but use SC-Met –Lys plates. You can streak out six matings per plate
reasonably well (6 plates per group). Incubate in 30oC incubator.
Assistants transfer plates to fridge on Friday if necessary to prevent background
growth of BY4741 derivates on – Met!
Day 3 (Tueday 2nd week): Test respiratory phenotype
When the diploid colonies are big enough to be picked, we test the mated mutants
for their ability to grow on medium containing only glycerol as a carbon source (glycerol
cannot be fermented, and therefore respiratory deficient cells will not be able to grow on
this medium). The Methionine selection is not very tight, so you may have to identify
the diploids by the larger colony size!
You need to have the original mutants on the plates as controls, as well as a positive
control so you have a reference for how well a respiratory competent strain should grow.
Streak out as below on SCGlycerol and YPD (as the growth control) for single colonies.
Mutant 1
BY4742
(respiratory
competent
control)
Mutant 1
OO
Mutant A
Mutant A
Mutant 1
OO
Mutant B
Mutant B
Mutant 1
OO
Mutant C
Mutant C
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YG 2015 P3
Repeat with mutants 2-6/A,B,C (six SCGlycerol and six YPD plates each group).
Incubate for 4-5 days in 30oC incubator (Glycerol is not a good carbon source, and yeast
grows much slower on glycerol plates than on YPD).
Day 4 (Wednesday 2nd week):
- Waiting…..
Day 5 (Friday 2nd week):
Waiting
Day 6 (Tuesday 3rd week): Scoring of growth
Inspect your plates. Make sure that the matings and mutant controls that do not
grow on SCGlycerol DO grow on YPD (if not, there is a general growth problem).
Note your results in the grid below (
mutants do not complement each other)
Mutant 1
Mutant2
+ = mutants complement each other; - =
Mutant 3
Mutant A
Mutant B
Mutant C
Determine complementation groups!!
Day 7/8/9: Nothing to do
Day 10 (Friday 4th week):
Review of results
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Mutant 4
Mutant 5
Mutant 6