Mystery strains
Today you become junior yeast geneticists
You will have to start thinking in order to carry out this genetic analysis.
Each pair of partner will receive the same four strains of yeast- (strains P, Q, R and S).
A strain may have between two and five mutations!
Mutations may contribute to the following phenotypes
Histidine auxotrophy
Uracil auxotrophy
Lysine auxotrophy
Leucine auxotrophy
Adenine auxotrophy
Colony color
In order to determine the genetic makeup of these strains, you may work together as a
class, sharing information and data with each other. Your ultimate goal is to provide a
genetic explanation for the inheritance of the phenotypes you see.
I suggest the following time-honored approach:
- identify the phenotypes of each of your strains. Do this by streaking out your strains and
then replica plating on different plates (YMD plates lacking one nutrient: -adenine or uracil or –histidine or –leucine), and observing their characteristics.
- Discuss with each other what you have found and see what sorts of matings you can
arrange, usually with some idea about how these matings will be informative.
- Once you have determined the behavior of the different mutations, you can think about
what you may expect when you sporulate your diploids (this means you will need to
dissect tetrads).
- unexpected things may emerge from some of these crosses, necessitating additional
complementation testing of the spore derived strains that arise from the tetrad dissections.
Feel free to discuss your results among yourselves. We will give you technical direction
and encouragement, but this is a puzzle designed to teach you by working through it
yourselves, using your observations and ideas. You can figure it out with what you have
on your plates!
The prep staff has provided you with a number of materials you may find useful. If you
feel you need something else, let me know and I will see if we can get it for you.
How to mate yeast:
Mix two strains of yeast on a plate on which they can both grow (YPD). Incubate at least
6-8 hours, better overnight or 24 hours. Patch or streak to selective medium on which
only the diploid and not either haploid, can grow. Test that the strain is diploid by
confirming that it sporulates. Multiple crosses can be done simultaneously by crossreplica plating.
Once you know the phenotypes of your strains.
You will need to plan time outside of class time to carry out this genetic analysis.
Mark the phenotypes of each strain up on the board, next to your name.
Discuss with each other what you have found and see what sorts of complementation
tests (matings) you can arrange.
Recall the point of complementation testing. It is to determine whether mutations that
cause similar or related phenotypes are in the same or different genes. If they are in the
same gene, complementation will usually not occur. If they are in different genes,
complementation will usually occur.
Does your strain have any similar phenotypes to those observed in any of the other
strains?
What will happen if you mate your strain with another strain that has a similar phenotype
and the mutations complement?
What will happen, if after the mating the mutations do not complement?
What will happen if the two strains you try to mate are the same mating type? Will you
be able to distinguish this outcome from non-complementation? How?
Make a plan to determine whether any of your mutants complement each other. Try to
think ahead.
If you streak cells out for mating on Tuesday, cross-stamp them on Thursday for mating
and then for selection on Friday then you should be able to score complementation next
Tuesday.
If you get diploids, DO NOT THROW THEM AWAY!!!!
The next step is sporulation and tetrad dissection.
TIPS ON TETRAD DISSECTION AND
MICROMANIPULATION OF YEAST CELLS
Adapted from notes by Cora Styles http://www.tiac.net/users/cstyles/Needle.htm
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Tetrad digestion: Spin down 0.2-0.5 ml of a liquid sporulation culture and aspirate-you
want a 10-15ul cell pellet.
Alternatively, add a barely visible tad of sporulated cells from the spore plate with a
sterile toothpick and twirl to scrape the cells off in the tube.
Add 50 ul of 1 mg/ml lytic enzyme, in sterile 1M Sorbitol, into the eppendorf and gently
pipet up and down with a P-20 to resuspend. Incubate at 37° for 4-7 minutes. Sometimes
you may need to incubate for longer or shorter times to get a good digestion. You should
have no problem with germination viability putting spores into the refrigerator for several
hours or even overnight (But I don't recommend it.).
After the incubation, put the tube on ice and gently add 0.2-0.8 ml of sterile water
to the tube, if necessary, mix by inversion. Leave on ice until ready to use. Spread onto
the bottom third of your dissection plate using a sterile loop. I usually spread 4-5 ul
worth of cells gently across the plate. It helps to draw a line across the bottom third of
the plate to keep track of where the cells are going. You can check for digestion by
putting the plate on the dissecting microscope and looking at the cells. You should see a
tight diamond shaped pattern of 4 spores. Anything that looks tighter will be difficult to
dissect. If you see groups of three tightly packed spores with a loosely attached fourth
spore nearby, ignore them, they may be fakes.
YPD is the standard media for dissecting ascospores. Some poorly germinating
mutants germinate better on GNA; some do worse. If you want to keep a plasmid in, I
still recommend dissecting on YPD, and printing the colonies to selective medium
afterward. Dissecting on selective medium (SC-X) will prevent full tetrads (4 spores)
from growing, and further, this medium is harder to pick up cells from (electrostatic
properties?).
Be selective about the plate you choose to work on. Is the agar surface parallel to
the edge of the lid all the way around? i.e. is it level? Level plates will show an agar
surface at the same height as the edge of the lid of the closed plate when viewed from the
side all the way around. The distance between the top edge of the bottom section of the
plate to the agar surface will be 7 mm. Try the squeeze test for moisture content: remove
the lid and hold the bottom between thumb and middle finger 180 degrees apart. Tilt the
surface to reflect light and squeeze firmly. A fresh plate will form tiny beads of water on
the agar just at the two pressure points. When the pressure is released, the droplets
disappear immediately. (This is also a good test for replica plates.)
Positioning your agar plate on the stage is like flying a helicopter, not a jet.
Landing, come over high, then descend. Taking off, go straight up, then depart. The
needle stands higher than the platform! See the section below for instructions on
positioning and adjusting the needle.
If you are having trouble picking up cells, check your agar plate. Some days I find
I'm chasing cells around and they just don't want to go on the needle. Then I realize the
plate I'm using has dried for only two days. I borrow a slightly older plate and my cells
now cooperate beautifully.
Before you pour your plates (for Saccharomyces) you can get rid of bubbles by
spritzing with alcohol.
The probability of contamination from the needle is insignificant. You can clean it
by plunging it once or twice into the agar plate if you wish. That's probably a good idea
just to clean debris off the tip anyway. Contamination is more likely to come from airborn mold spores and bits of lint and lab air detritis which the static electricity attracts
when you place your plate on the microscope. If you have compressed air in a can, you
might want to blow out your dissecting chamber. Contaminating colonies can be
"surgically removed" from your plate conveniently with the thin flat blade of an artist's
palette knife dipped in alcohol and flamed handy tool. (Cut all the way down to the
bottom of the plate and lift out a small chunk of agar with the offending contaminant.) If
contamination is really bad, try to set your microscope up somewhere else.
Manipulation technique: When the flat surface of the needle touches the surface of the
agar, a thin layer of water forms between them and surface tension binds the surfaces
together. You can see a dark ring suddenly appear surrounding your needle. Be sure to
focus sharply on the cells or on your needle touching the agar. When the needle is pulled
away, the water connection stretches and finally breaks. A cell in the water will either
end up on the needle or on the agar. Other times, the cells cling to the side of
the needle and are deposited at the next stop.
If cells don't leave the agar surface, you have to apply some additional technique.
Cells can be coaxed on to the needle surface better if there is movement east-west or
north-south at the same time as pulling away, which results in an arc-like motion. Try
different maneuvers to see what works best for you. Sometimes pulling, sometimes
pushing, sometimes a rather abrupt snatch. You may find a "sweet" side of your needle.
Stick with it.
Put cells down gently. Approach with caution because the surface at the new
position may be out of focus. You simply can't work out of focus. If the cells don't come
off the needle, try positioning the needle just short of the agar and tapping on the table or
stage to make the needle bounce gently on the agar, and snatch the needle away quickly
to avoid re-picking them up again. This maneuver also separates ascospores from each
other.
Cells vary in the amount of handling they can survive. Ascospores are very
durable. Stationary phase cells and zygotes are quite tough, too. Log phase cells are
sensitive. My personal rule for log phase cells is that if I can't pick up the cell on the first
attempt, I leave it and go on to another. I don't want to waste my time carrying around
dead bodies.
Preparing to Dissect: Positioning and Adjusting the Needle:
Look at the micromanipulator (also, refer to the figure ). It is attached to the
stage, so it will move up and down with the stage as it is focused. The plate holder, the
aluminum block on top of the stage with the semi-circular cut-out, can be moved over the
stage with the X-Y adjustment knobs that hang below and to the right of the stage.
The dissection needle support shaft is attached to the tool holder. The ideal
position of the needle support shaft is parallel to the surface of the stage. This allows the
greatest up-down range. The bend in the shaft is a right angle, thus your needle will be
perpendicular to the agar in this plane. Adjust the tilt of the shaft by turning the x-axis
adjustment knob.
Normally, the needle will already be positioned so that it is in the middle of the
visual field-all you have to do is position the needle vertically so that it can reach the
agar. To begin with, raise the joystick and then position the needle so that it is close to
the stage. Put your plate on the plate holder-agar side down. Look at the plate from the
side and note the position of the needle with respect to the agar. You want it to be 1-2
mm from the agar. To raise or lower the needle with respect to the stage, turn the z-axis
control knob clockwise to raise the needle, turning counter-clock wise will lower it. Be
careful! One of the most common ways needles are broken is when the
micromanipulator is lowered instead of raised, resulting in the needle being snapped off
by the stage.
Now look through the objectives and focus onto the surface of the agar. The
easiest way to do this is to focus onto cells on the plate. Look for the circular shadow of
the needle. If you can't see it, carefully turn the z-axis knob clockwise. Once you can see
the needle, turn the z-axis knob counter-clockwise about 1/4 turn. Now gently lower the
joystick until the needle touches the surface of the plate. Note the position of the
joystick: ideally it will be paralell to the ground or even pointing slightly down.
You can adjust the amount of movement from the joystick that is transmitted to
the needle by turning the joystick around its long axis. The split ball joint will open or
close. When the ball joint is closed, very little movement is transmitted to the needle and
when the ball joint is open, more of the joystick movement will be translated to the
needle.
Centering the needle. If the needle is out of position, or if you need to mount a new
needle, follow these directions to re-center it. Position the needle roughly in the center of
the lens by looking sideways at the objective. Use the low-power lens (2x) useful for
rough centering. Fine tune the centering by looking through the higher power objective.
Bring it close to the needle to be sure to see it. Once found, back off till the tip is in
focus. North-South (i.e. closer or farter away from you) adjustments can be made by
turning the tool tilting screw to bring the needle into the center of your visual field.
Tilt adjustment: Focus on the surface of the needle tip and then focus downward. Does
the bore of the needle shaft change position more than just a little? North-south tilts can
be corrected by rotating the support shaft. (Take into account that the visual image is
inverted.)
Trouble-shooting:
"I know the needle is centered, but I can't see it when I put my plate on."
The needle is too short to reach the agar. The tell-tale sign is that the support rod
strikes the edge of the plate when the operator tries to bring the needle up to see it.
If the support rod sections are taped together, perhaps the tape is too thick andit
hits the edge of the plate.
"I can't focus on my cells."
The agar is too thick and exceeds the focal distance of the objective. The
objective pushes against the bottom of the plate as the operator tries to focus.
"The cells which I picked up have disappeared. Did they fall off?"
Cells do not fall off into outer space. They are somewhere, either on the needle or
on the agar. If they are on or near the surface of the needle, continued patience
will coax them off. They might be down on the side of the shaft. These just won't
come off. If you decide to give up on this tetrad and go on to another, be sure you
start with a clean needle. In an area away from the ascospore grid, plunge the
needle into the agar a few times. You may even drag the needle across the agar,
too. Agar doesn't break needles. Plastic does.
"My needle seems to swallow cells. I'm always losing cells."
Check to see the needle is perpendicular.
The needle surface may be dirty or defective. With repeated use, needles build up
a film of agar on their surface. Remove this by soaking/plunging the tip in agar
and leaving it there a few minutes. Then plunge some more times and drag it
across the agar surface. Can you see a trail of something clinging to the tip? Try
to get rid of it.
Defects of the needle are usually visually observable, especially if it transmits
light. There may be a spur or a chip on one edge. Such a needle needs to be
replaced.
Tetrad dissection
By now you should have some information concerning which strain mates with which
and complements each other.
Once you know the complementation behavior of your strains:
Mark the complementation behavior of your strains on the board
One of the first things to determine is whether the phenotype you are studying is caused
by one or two genes.
Discuss with each other what you have found and see what sorts of segregation data will
be needed.
Remember that just because a strain fails to complement another does not mean that
either strain has one and only one mutation.
In yeast, tetrad dissection is an important component of genetic analysis.
What kinds of tetrads are there?
What kinds of tetrads will you get if a phenotype is caused by a mutation in a single
gene?
What kinds of tetrads will you get if a phenotype is caused by a mutation in a single
gene?
What kinds of tetrads will you get if the phenotype is caused by either or both of two
unlinked mutations?
If you can separate such mutations, how can you keep track of them?
A second important thing is to determine whether and how mutations are linked. Recall
the point of linkage testing by tetrad dissection. It is to determine whether two mutations
that cause observed phenotypes can be separated by recombination, and what their
recombination distances might be. If they are the same or overlapping mutations, no
recombination can be observed. If they are different mutations in the same gene,
recombination will be very infrequent. If they are in different genes near each other on
the same chromosome, they may display linkage, whereas distant genes on the same
chromosome, and genes on different chromosomes will segregate independently.
How does linkage affect the kinds of tetrads observed?
What kinds of tetrads will you get if the two genes are tightly linked?
What kinds of tetrads will you get if the two genes are unlinked?
What kinds of tetrads will you get if the two genes are loosely linked?
Micromanipulator
There will be a quiz on tetrad analysis.
There will be a demonstration of the micromanipulator and everyone needs to try it.
Practice picking up and moving yeast cells around with the micromanipulator. You will
need to build up your skill and patience in this area in order to dissect tetrads
successfully.
Spread some yeast on a thin dissection plate and try to pick up a single cell at a time and
put it down on the plate somewhere else without killing it or breaking the needle.
Someone will demonstrate the use of the micromanipulator. There are several
adjustments on the device, which must be attended to. Everyone gets to try. If there are
tetrads to dissect, we can try those too, but that is a little trickier and it helps to first get
the feel of the joystick.
Analysis of dissected tetrads
Hopefully by now you have all successfully dissected some tetrads. You are wondering
what to do next. Here are some suggestions.
1
Make sure you keep track of the spores in each tetrad. Make sure you know which
spores go with which!!! These are the products of a single meiotic event.
Score all the phenotypes you observed in the haploid strains that gave rise to the diploid.
Note any new phenotypes that arise in the progeny of the diploid that were not detected in
the previous generations. For example: 3:1 segregation.
2
Observe the segregation pattern within each tetrad, one marker at a time!
Is there a pattern?
For a phenotype caused by a single mutation, two spores within a tetrad should display
one phenotype and two should display the other.
If the phenotype is caused by a combination of two genes, the pattern will be more
complex. If each gene independently causes the same phenotype as the other and as the
double mutant, and the genes are unlinked, then there will be many (~4/6) tetratype
tetrads, which will have three spores showing the mutant phenotype (two spores, one
with each single mutation and one double mutant spore) and one wild type spore (no
mutations). Set these more complex segregation patterns aside for the moment and
consider as many simple cases as you can.
3
For the markers that appear to segregate as single genes, consider them in pairs to
determine whether they are linked or unlinked.
We now know from our complementation analysis that there are three different
genes in our strain set that give rise to histidine auxotrophy. In the case where a
diploid has his- mutations in two different genes, the diploid will be his+, but many
of the spores will be his-. Consider a cross where black is his+ and white is hisQUIZ 1: linked or unlinked?
QUIZ 2: linked or unlinked?
In this example, we consider tetrads for the oval/round and the black/white phenotypes,
each of which segregates as a single gene.
Tetrad examples
Situation1:
All PD (tetrad #s 1, 2, 3, 4, 5, 6)
Conclusion: Tightly linked
Situation2:
1PD (tetrad #5)
1 NPD (tetrad #3)
4 TT (tetrad #s 1, 2, 4, 6)
Conclusion: unlinked
Note that loose linkage will generate an intermediate pattern with PD>NPD with some
TT. Use the equation to estimate linkage.
Situation3:
We know from our complementation analysis that there are three different genes in our
strains that give rise to histidine auxotrophy. In the case where the diploid has hismutations in two different genes, the diploid will be his+ (complementation), but many of
the spores will be his-.
Consider a cross where black is his+ and white is his- caused by either of two different
unlinked his mutations:
Can you write the HIS genotype for each of these spores? The solution involves
observing the complementation behavior of the spores after they are mated to tester
strains carrying one or other his- mutation. If a spore makes a his+ diploid after mating to
hisA, it must not be hisA-. Therefore it must be hisB-. Spores that do not make his+
diploids after mating to either hisA or hisB must be hisA-, hisB- double mutants.
Once we get a set of tetrads that we trust, you will need to analyze them as we have done
with this exercise, taking each phenotype as it comes pairwise in each cross. Because the
crosses are different, you will see contradictory things if you try to compare the results
from two different crosses too soon. Thus analyze each cross separately first, then
compare the results from the different crosses