McClean: Tetrad Dissection

Overview

This protocol describes dissection of yeast tetrads. In our lab, we primarily use tetrad dissection for constructing strains for genetic and biochemical experiments.

An accomplished yeast biologist can dissect a plate of spores in 20 minutes; for a beginner, 2 hours is not unusual. Tetrad dissection is a learnable skill. Your initial attempts will likely be frustrating. If you persevere, you will be richly rewarded by your new ability to wield one of the most powerful tools in the yeast geneticist’s toolbox. When you first learn to do tetrad dissection, make sure to ask for help from someone in the lab who is experienced at doing it! It really helps to have someone with a practiced eye point out what a well-digested culture looks like, what a tetrad looks like under the dissecting scope, etc.

Materials

• β-glucuronidase (Sigma G7770, Stored in 4°C refrigerator. Comes as an aqueous solution in ~1.0 M ammonium sulfate with 3 mM sodium azide as preservative.)
• Sporulated yeast culture
• Sterile Water
• "Dry" YPD dissection plates
  • Everyone has a particular way that they like their plates for tetrad dissection. You are basically aiming for dry and level. To make dissection plates, add 25 ml YPD with a plastic strippette to plates on a very level surface. Once solid, invert. Let dry at room temperature for ~3 days. Bag. These plates are best after aging for a while.

Protocol

Digestion

The spores are contained in a specialized cell wall called an ascus. In order to be able to separate the spores, the ascus is digested with the enzyme B-glucuronidase. Ideally, the ascus is digested just enough that the spores can be separated but not so much that the spores fall apart entirely.

1. Spin down 250 ul of sporulated culture.
2. Resuspend in 250 ul sterile water.
3. Mix 17 ul washed culture with 3 ul B-glucuronidase (can vary by strain). Set up three tubes like this – you will stop them at different time points to check digestion.
4. Let sit at room temperature 20, 30, and 40 minutes. Stop the digestions as in step 5. (The ideal timing for digestion varies by strain, culture, and ambient conditions – if you are having trouble with your dissections, you may need to try less or more time. Check with an instructor for their opinion if you are having trouble with your dissection.)
5. Gently add 100 ul water. The goal is to suspend the cells without breaking up the tetrads. Tap the tube gently to mix.
6. Transfer 5 μl of each test digestion to a slide and cover each with individual coverslips. Check for digestion under the upright microscope (see next section on appearance of tetrads).
7. Mark the plate at opposite edges to indicate the center of the plate (see following diagram).
8. Resuspend the digested cells from your chosen timepoint. by gently tapping or pipetting (they will probably have settled while you were looking at the digestions under the microscope). Drip 20 μl down the imaginary center line you just indicated, beginning from one edge.
9. Let the digested culture fully absorb into the plate.
10. Dissect (see dissection section). If you dissect on a different day, you must begin with a fresh plate and set up a new dissection. The sporulation cultures are good for weeks, though the spore viability may start to decrease.

Appearance of Tetrads

Before digestion, tetrads are held tightly in a tetrahedron and it is often difficult ot see all four spores at once. After digestion they relax into a diamond shape.

How to distinguish a tetrad from two budded cells adjacent to each other? 1) Generally, the spores of a tetrad are smaller than a budded cell. 2) Tetrads, if moved gently, will remain intact while two adjacent budded cells will not. 3) The four spores in a tetrad are often very similar in size, while a budded cell usually has a large mother cell and a smaller bud.

4 spores of a tetrad. After digestion, most tetrads should have this appearance. Tetrad with 3 spores visible in one focal plane and 4th spore visible in a second focal plane. Before digestion, many tetrads may have this appearance. The Goal of your Dissection

Dissection

The dissecting scopes have a specially designed stage with “clicks” you can feel in both the x and y direction that allow even spacing of tetrads. The micromanipulator is used to control the position of the needle.

1. Drop the needle to its lowest position by raising the arm of the micromanipulator (yes, this seems somewhat counterintuitive!!) 2. Move the turret of the scope so that there is no objective lens over the stage. This will give you room to position the plate. 3. 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! 4. Position the plate on the stage so that the two marks you made line up with the horizontal axis of the plate. 5. Swing the objective into position, and move the stage so that the objective is centered over the swath where you dripped the digested sporulation down the plate. 6. Focus on the cells sitting on the surface of the plate (you will be focusing through the agar into the cells sitting on the surface). 7. Pick tetrads. i. Move the microscope stage so that individual tetrads with a clear zone around them are visible. Raise the needle (by lowering the arm of the micromanipulator) until you can see it. ii. Pick up the four spores with the microneedle. I like to place the needle very near to (but not touching) the spores, and if they are a genuine tetrad they will be sucked by surface tension as a group toward the needle. iii. Place them on the agar in a click position at least 5 mm from the stripe of b-glucoronidase-treated cells. Note the position on the mechanical stage. iv. To break apart the four spores, place the needle so that it barely touches the tetrad then gently tap the base of the microscope or table beside the microscope. This vibrates the needle enough to break apart the tetrad without jabbing the needle into the agar. v. Pick up three spores. Move the stage 5 mm (one click) further away from the streak and deposit the three spores. vi. Pick up two spores. Move the stage 5 mm (one click) away from the streak and deposit the two spores. vii. Pick up the remaining spore. Move the stage 5 mm (one click) and deposit the remaining spore. viii. Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate. ix. Remove the YPD plate from the stage, taking care not to break the microneedle. 8. Incubate the plate for 2-3 days at room temperature. We will test the phenotype (auxotrophic or not) and mating type of your spores.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

1. *Megan N McClean When I can't age my dissection plates on my bench for a few days, I will stick them in the 30°C or 37°C warm room the morning of the day I dissect to dry them out a little bit. It is absolutely infuriating, if not impossible, to try dissection on plates that are wet.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

1. *Megan N McClean Different tetrad dissection protocols call for using different enzymes to digest the ascus wall. Our protocol uses a β-glucuronidase from Sigma (G7770) which is a mixture of enzymes derived from Helix pomatia (the Roman snail). Zymolyase, another commonly used enzyme, consists mostly of β-1,3-glucan laminaripentaohydrolase. It hydrolyzes glucose polymers at the β-1,3-glucan linkages releasing laminaripentaose as the principal product. β-glucuronidase catalyzes hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides (also referred to as glycosaminoglycans).

References

Adapted from Maitreya Dunham's protocol (http://dunham.gs.washington.edu/sporulationdissection.htm) and the Botstein lab protocol (http://www.princeton.edu/genomics/botstein/protocols/Sporulation_and_Tetrad_Dissection.pdf)

Contact

• Megan N McClean 14:01, 20 July 2011 (EDT)

or instead, discuss this protocol.