Effektiv Sporulation af<em> Saccharomyces cerevisiae</em> I en 96 Multiwell format
Summary
Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.
Abstract
During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.
Introduction
Sporedannelse i knopskydende gær er blevet undersøgt for at give indsigt i mange aspekter af biologi, herunder kontrol af kromosom segregering under meiose 1, mekanismer for genetisk rekombination 2, kontrol med udvikling af cellesignalerende 3, ernæringsmæssige kontrol med udvikling 4, den transkriptionelle regulering af udvikling 5, og undersøgelsen af sporedannelse 6. Spore dannelsen indeholder et nyt celledeling begivenhed involverer dannelsen af nye membran rum inden moderen celle efterfulgt af aflejring af en beskyttende spore væg 6. Disse undersøgelser, der undersøger sporulerende celler tager ofte fordel af det hastigt sporedannende gærstamme SK1, som kan gennemgå processen med sporulering i omkring 24 timer i en relativt effektiv måde 7,8. Selvom optimering af sporulering betingelser for knopskydende gær er blevet beskrevet 9-13, disse forsøgs undersøgte sporedannelse på faste medier eller i større målestok flydende kulturer hvor sporedannelse udføres under anvendelse kultur rør eller kolber.
Her beskriver vi en fremgangsmåde til sporedannende gær i en 96 flerbrøndsplade format. Vi finder, at denne metode, beluftning er kritisk for synkron og effektiv sporulering, og har udtænkt teknikker til at sikre tilstrækkelig sporulering et lille volumen brønde format. Sporedannende i en 96 flerbrøndsplade format giver mulighed for celler, der skal screenes ved hjælp af høj-throughput teknikker og reagenser optimeret til en flerbrøndsplade format, såsom screening for højt kopiantal suppressorer bruger flisebelagt bibliotek 14-16.
Protocol
Representative Results
Discussion
Her præsenterer vi en protokol for sporedannende SK1 gær i en 96 brønde format. Beluftning er nøglen til effektiv sporulering, der kræver brug af enten en omrører eller en glasperle i hver brønd. Når celler sporuleret i en 96 flerbrøndsplade i en rysteinkubator uden enten en vulst eller en omrører, gør cellerne ikke danne sporer effektivt. Kun en lille stigning i sporedannelse effektivitet ses når celler sporuleret uden enten en vulst eller en omrører i en rysteinkubator, sammenlignet med at være ved 30 °…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af en Joseph P. Healey bevilling fra University of Massachusetts Boston (LSH) og R15 GM86805 fra NIH (LSH). SMP understøttes delvist af en Sanofi-Genzyme Fellowship ved University of Massachusetts Boston.
Materials
| Nunc 1.3 ml DeepWell Plates | ThermoScientific | 260251 | Used for sporulation |
| Nunc 2.0 ml DeepWell plates | ThermoScientific | 278743 | Used for presporulation growth, step 1.2.3 |
| 3 mm glass bead | Fisher | 11-312A | Used for sporulation |
| 5 mm x 2 mm stir bar, pack of 12 | Fisher | 14-511-82 | Used for sporulation |
| 96 well frogger | V&P Scientific | VP407 | needed for step 1.2 |
| library copier | V&P Scientific | VP381 | needed for step 1.2; to be used with the frogger |
| rectangular petri dish | ThermoScientific | 264728 | needed for step 1.2 |
| Bacto Peptone | BD | 211677 | needed for media |
| Yeast Extract | BD | 212750 | needed for media |
| Bacto Agar | BD | 212750 | needed for media |
| Dextrose | Fisher | D16-3 | needed for media |
| Potassium Acetate | Fisher | P171-500 | needed for media |
| Glycerol | Fisher | G33-500 | needed for media |
| Black 96 well glass bottom plate | MatTek | PBK96G-1.5.5-F | needed for step 2.4 |
References
- Marston, A. L. Chromosome segregation in budding yeast: sister chromatid cohesion and related mechanisms. 유전학. 196 (1), 31-63 (2014).
- Keeney, S., Lange, J., Mohibullah, N. Self-organization of meiotic recombination initiation: general principles and molecular pathways. Annu. Rev. Genet. 48, 187-214 (2014).
- Granek, J. A., Kayikci, O., Magwene, P. M. Pleiotropic signaling pathways orchestrate yeast development. Curr. Opin. Microbiol. 14 (6), 676-681 (2011).
- Broach, J. R. Nutritional control of growth and development in yeast. 유전학. 192 (1), 73-105 (2012).
- Winter, E. The Sum1/Ndt80 transcriptional switch and commitment to meiosis in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 76 (1), 1-15 (2012).
- Neiman, A. M. Sporulation in the budding yeast Saccharomyces cerevisiae. 유전학. 189 (3), 737-765 (2011).
- Padmore, R., Cao, L., Kleckner, N. Temporal comparison of recombination and synaptonemal complex formation during meiosis in S. cerevisiae. Cell. 66 (6), 1239-1256 (1991).
- Liti, G., et al. Population genomics of domestic and wild yeasts. Nature. 458 (7236), 337-341 (2009).
- McCusker, J. H., Haber, J. E. Efficient sporulation of yeast in media buffered near pH6. J. Bacteriol. 132 (1), 180-185 (1977).
- Codon, A. C., Gasent-Ramirez, J. M., Benitez, T. Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker’s yeasts. Appl. Environ. Microbiol. 61 (2), 630-638 (1995).
- Elrod, S. L., Chen, S. M., Schwartz, K., Shuster, E. O. Optimizing sporulation conditions for different Saccharomyces cerevisiae strain backgrounds. Methods Mol. Biol. 557, 21-26 (2009).
- Börner, G. V., Cha, R. S. Analysis of yeast sporulation efficiency, spore viability, and meiotic recombination on solid medium. Cold Spring Harb. Protoc. 2015 (11), 1003-1008 (2015).
- Börner, G. V., Cha, R. S. Induction and analysis of synchronous meiotic yeast cultures. Cold Spring Harb. Protocols. (10), 908-913 (2015).
- Jones, G. M., et al. A systematic library for comprehensive overexpression screens in Saccharomyces cerevisiae. Nat. Methods. 5 (3), 239-241 (2008).
- Fleming, M. S., Gitler, A. D. High-throughput yeast plasmid overexpession screen. J. Vis. Exp. (53), e2836 (2011).
- Paulissen, S. M., Slubowski, C. J., Roesner, J. M., Huang, L. S. Timely Closure of the Prospore Membrane Requires SPS1 and SPO77 in Saccharomyces cerevisiae. 유전학. , (2016).
- Amberg, D. C., Burke, D., Strathern, J. N. . Methods in Yeast Genetics: A Cold Spring Harbor Labooratory Manual. , (2005).
- Parodi, E. M., Baker, C. S., Tetzlaff, C., Villahermosa, S., Huang, L. S. SPO71 mediates prospore membrane size and maturation in Saccharomyces cerevisiae. Eukaryot. Cell. 11 (10), 1191-1200 (2012).
- Nakanishi, H., de Los Santos, P., Neiman, A. M. Positive and negative regulation of a SNARE protein by control of intracellular localization. Mol. Biol. Cell. 15 (4), 1802-1815 (2004).
- Huang, L. S., Doherty, H. K., Herskowitz, I. The Smk1p MAP kinase negatively regulates Gsc2p, a 1,3-beta-glucan synthase, during spore wall morphogenesis in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA. 102 (35), 13431-13436 (2005).
