Mating, Spore Dissection, and Selection of Diploid Cells in Schizosaccharomyces japonicus
- 1Radiation Biology Center, Kyoto University, Yosida-Konoe-Cho, Sakyo-Ku, Kyoto 606-8501, Japan;
- 2Microbial Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
- ↵3Correspondence: hniki{at}nig.ac.jp
Abstract
Haploid yeast cells mate to form heterozygotes and subsequently undergo meiosis to form spores. This process can be used to produce gene combinations and variants that are useful for genetic analysis. For example, these spores can be used to generate double mutants or to measure genetic distances in a mutational analysis. Here, we describe mating and spore dissection procedures for Schizosaccharomyces japonicus cells. Although the overall procedures resemble those used in Schizosaccharomyces pombe, some differences exist, including the use of EMM2 medium without nitrogen (EMM-N) for mating and the shorter incubation time of 16–20 h for S. japonicus cells. Furthermore, the S. japonicus zygotes produce eight spores and thus require an “octad” analysis.
MATERIALS
Reagents
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Prepare EMM-N liquid medium. (EMM-N is EMM2 medium without nitrogen.)
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Prepare EMM2 agar plates without adenine.
Schizosaccharomyces japonicus strains
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Strains are available from the web home of the S. japonicas bioresource center, JapoNet (http://night.nig.ac.jp/labs/MicroGen/japonet/).
ade6-domK and ade6-domE mutant strains (for selection of diploid cells; see Steps 9–12)
Heterothallic cells of opposite mating types (for induction of meiosis; see Steps 1–8)
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Prepare liquid medium and 2% agar plates. If necessary, the YE medium can be supplemented with 0.1 mg/mL adenine and 0.1 mg/mL uracil.
Zymolyase 100 T (1 mg/mL)
Equipment
Heat block at 37°C
Micromanipulator
Sonicator (optional; see Step 11)
Tabletop centrifuge
Test tubes (18-mm)
Vortex
Water bath incubator with shaker at 30°C
METHOD
Induction of Meiosis by Nitrogen Starvation
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Nitrogen deficiency is effective for inducing sporulation. The best way to sporulate is by using liquid medium. Portions of this procedure were previously described in Furuya and Niki (2009).
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1. Inoculate each mating type of heterothallic cells in 3 mL of YE liquid medium in 18-mm test tubes, and shake overnight at 30°C.
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The cells must be saturated the next morning.
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2. Centrifuge the cells to harvest (1100g for 1 min in a tabletop centrifuge at room temperature). Remove the supernatant.
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3. Resuspend cells in 0.5 mL of EMM-N liquid medium. Vortex well.
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4. Prepare a new test tube (18-mm diameter) with 0.5 mL of EMM-N liquid medium, and add 0.05 mL of both mating types of heterothallic cells.
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5. Incubate the mixture of heterothallic cells for 16–20 h at 30°C.
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It is not necessary to shake the mixture in the test tube; the tube can simply be left standing for incubation. Overnight incubation in EMM-N liquid medium at 30°C provides a sufficient number of spores to work on.
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6. Check for sporulation by microscopy.
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A dumbbell-shaped ascus includes eight spores.
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See Troubleshooting.
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7. Harvest and centrifuge the cells in a tabletop centrifuge (1100g for 1 min at room temperature). Add 0.1 mL of 1 mg/mL Zymolyase 100 T to the cell pellet. Place in a heat block for 5 min at 37°C.
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Fresh asci are hard to dissect. Digesting the ascus wall with Zymolyase 100 T helps to subsequently release the eight spores for dissection using the micromanipulator needle. If the mixture is standing at room temperature for an extended period of time, the asci are autonomously lysed so that the spores are released. Spore viability is maintained for 1 wk at 4°C.
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8. Spread the ascospores onto YE 2% agar plates for dissection. Perform spore dissection using a micromanipulator.
Selection of Diploid Cells
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The ade6-domK and ade6-domE alleles show interallelic complementation when they are simultaneously expressed in the same cell. As with S. pombe ade6-M210 and ade6-M216, this facilitates the construction of diploid cells (Furuya and Niki 2011).
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9. Mix the two ade6 mutants, one of each mating type, following Steps 1–4.
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10. Incubate the mixture for 5 h at 30°C.
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Because heterozygotes are formed in 4–5 h, cell aggregation, namely sexual flocculation, can be seen in the tube.
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11. Spread cells in the mixture onto EMM2 agar plates lacking adenine.
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If necessary, aggregated cells can be gently sonicated.
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12. Check for diploid cells under a microscope.
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The diploid cells are twice as large as the haploid cells.
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TROUBLESHOOTING
Problem (Step 6): Some of the selected clones of diploid cells lose their ability to sporulate.
Solution: Multiple clones of diploid cells should be isolated and tested.
Footnotes
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From the Fission Yeast collection, edited by Iain M. Hagan, Antony M. Carr, Agnes Grallert, and Paul Nurse.
