P2 2015
One of the major reasons why yeast has been such a successful model organism is its ability to grow in haploid and diploid life cycles. The possibility of creating mutants in haploid cells and the opportunity to analyze and combine different mutations via mating of mutants or mating back to the wild type allowed for genetic dissection of metabolic pathways long before recombinant methods were established. For example, the position of mutations within a pathway can be determined via epistasis analysis . In combination with biochemical methods, genes coding for proteins with certain biochemical properties
(enzymatic activity, etc.) could be characterized long before it was completely clear what exactly a gene was. In addition, the physical location and distance of genes can be inferred by linkage studies .
In this project, you will practice
1.
How to carry out matings (it’s very easy)
2.
Select for diploids – good practice for streaking techniques to obtain single colonies from a culture stock
3.
How to dissect a tetrad – this requires some practice, but it is fun once you get the hang of it. While you may not ever again dissect yeast tetrads, you can regard this as your first encounter with a micromanipulator (in this case, our tetrad dissection microscope).
4.
How to extract information about a gene or (a) mutation(s) by the way they segregate during meiosis (we are analyzing the meiotic products (=tetrads) from a diploid)
5.
st
As you hopefully remember from the lecture, haploid yeast, like humans, comes in two flavors (or more precisely mating types ), a and

. An a strain will only mate with

and vice versa. Matings do not occur between two a cells or two
 cells. This is due to the mating pheromones and receptors made in the different cells types ( a cells make a -factor pheromone and
 factor receptor;

cells make the opposite types. We will be working with the following strain:
BY4742(α)
We will mate this strain to:
W1536 5B ( a ) and W1536 8B (

). These strains are isogenic (i.e. their genetic background and marker mutations are identical) with the exception of their mating type.
I will only give partial information for the genotype: Both are Lys
+ will examine the Ade
–
, Trp
-
and Ade
-
. We phenotype of the W1536-derived strains more closely during this practice.
Which mating should yield in diploids?
We will also mate:
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P2 2015
BY4742
 pdr13 (isogenic to BY4742, but carrying a replacement of the pdr13 gene by the KANMX kanamycin resistance cassette) to mutant htd2-4 – a derivative of W1536 5B that carries a mutation in a mitochondrial dehydratase that renders the strain respiratory deficient. The relationship between these mutations will be examined.
The mating procedure:
1. Prepare the YPD plates: With a waterproof pen, label the top of your plate like this:
W1536 5B
BY4742

 pdr13
W1536 8B
htd2-4
Also put a date and your initials on the plate, and note what type of plate it is. It may seem silly at this moment, but once you start dealing with a lot of different types of plates and share an incubator with other people, this procedure becomes essential. If you don’t do this minimal kind of labeling, you will confuse your own plates (plate type as well as when you streaked out a certain plate; or you’ll mix them up with other peoples plates). This is just good lab practice, and believe me, it will save you a lot of time, anger, confusion and desperation if you make it a habit .
Mating: Start with BY4742: Take a toothpick-tip (fat end) full of cells, make a streak in the BY4742 field and a streak in each of the mating (OO) fields (always use sterile toothpicks!!!). Then, take a toothpick full of W1536 5B cells, make a streak in the
W1536 5B field and the rest of the cells right on top of the BY4742 cells in the adjacent field, mixing the cells by “stirring” them together. Don’t use too much pressure, because you do not want to break the agar. (We will demonstrate)
Do the same with W1536 8B (of course in its designated fields).
Streak the BY4742
 pdr13 cells in their designated field and in the mating field, then do the same with mutant htd2-4 and mix.
That’s it. Now we just have to put the cells into a warm (30 o
C) and dark (optional) place o/n.
Mating can take place in as little as 6-8 hours.
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P2 2015
After the cells have mated, we have to have a way of distinguishing between haploid and diploid cells. In our case, we will follow the Lys and Trp markers. Only the diploid cells, which have the sets of genes from both parentals, will be able to grow on medium lacking both tryptophan AND lysine. The haploid parentals will not be able to grow, as they will lack the ability to make either lysine ( BY4742 ) or tryptophan (W1536
5B or 8B). If the mating did not occur (because the mating type didn’t match), there will be no survivors on the plates.
Label the SC – Trp, -Lys plates like this: htd2-4
W1536 5B
Inside-Out streak
BY4742
 pdr13
BY4742
W1536 8B
Christmas tree streak
Take a toothpick (fat end) tip full of mated cells and streak for single colonies in the large sector of the plate. You can do this in several ways. The easiest is probably the
“Christmas Tree” type streak (see picture). You first make a streak on the bottom, then you turn around the toothpick and make a short perpendicular streak towards the inside of the plate. The third streak is carried out with the skinny end of a fresh toothpick, touching the end of the second streak (just on the edge) and then pulling from side to side and towards the center of the plate. The more distance you cover, the better your separation will be. The “inside out” streak works as following: First, you make a streak of cells from your mating on the INSIDE of the plate with a fat end toothpick. Then you draw out the cells (again from the edge of the first streak) side to side towards the outside of the plate with the skinny end of a fresh This streak is harder to do and needs more practice, because you have to PUSH the toothpick, which much more easily can result in scraping or penetrating the agar. The advantage is that at the end of the streak, when there are only few cells on the toothpick, you get your best “resolution”, as there is more travel for the toothpick.
It is also important to streak out the parentals as a control to make sure they really do not grow on this media. However, because we don’t expect them to grow and we don’t need very accurate single colony separation, we will only streak in a small sector.
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P2 2015
Place the plate in the 30 o
C incubator. Colonies should appear until next week
(usually 2-3 days).
Assistants check the plates on Friday and Monday; transfer to fridge if colonies are big enough)
nd
By now, colonies should have appeared on the plates. As these originated from cells that were able to both make lysine and tryptophan, we can assume that they are diploids created by the mating of cells from our two parental strains. Check the streaks of the parentals to make sure there is no growth there (otherwise we have a problem!).
Alex’s group (the others will do this on Friday:
Every student pick two colonies from each of the mating/diploid streaks and streak them on sporulation (f-spo) plates.
Sporulation occurs on media lacking nitrogen and containing a poor carbon source (usually acetate).
The simplest type is potassium acetate, 10g/L, yeast extract 1g (or 2.5g)/L, glucose 0.5 (or 1g) per liter. The medium we are using is more complicated (it is more like synthetic complete medium). We call it “fancy sporulation (f-spo) medium”. It takes more effort to make it, but we find the sporulation for the diploids we are working with is more efficient on these plates than any other plates. The f- spo medium is a synthetic medium that has only a limited amount of amino acids and vitamins.
Divide plate in four zones and streak out diploids. No need to streak for single colonies – patches are fine!
Put the plates in the 30 o C incubator for 3-5 days .
nd
Waiting…
nd
The other groups streak out their diploids for sporulation (see above)
Alex’s group: Checking for spores/tetrad dissection (others will do this next week):
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P2 2015
Pipet 50
 l of water on a microscope slide. Scrape a loopful of cells from the patch of sporulated diploids and suspend in the water puddle. Put on a coverslip and examine the cells under the microscope (60x magnification). You should recognize the tetrads by their unique shape: four spherical balls forming a tetrahydral “pyramid” (Left example)
Sometimes, you can only see three spores because one of the spores is out of the plane of focus (right example).
If you have spores, we will carry out a tetrad dissection. Follow the instructions of the “Ascus Dissection” procedure by Carl Saunders-Singer. We will help you individually.
When you are done with the dissection, put the plate in the 30 o C incubator for 2-3 days.
When the spores have grown out to large enough colonies (≥2mm), inspect them visually. Note down appearance, esp. color.
Replica plate the colonies on SC-Ade plates.
Students that dissected the htd2-4/pdr13 diploid also replica plate:
on SCG (glycerol) plates
on YP-Geneticin plates
(We will show how it’s done)
Transfer the plates in the 30 o C incubator for 2-6 days, depending on which diploids you are testing.
th
What do you notice about the KANMX (geneticin resistance) phenotype and the respiratory deficient phenotype of the colonies? What does that tell you about the genes responsible for these phenotypes?
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P2 2015
What do you notice about the Ade- phenotype and colony color? What does it tell you about the Ade- mutation. Can you guess in which gene the mutation is? Describe your observations also using terms like tetratype (TT), parental ditype (PD) and nonparental ditype (NPD).
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