1. Mechanisms of Dormancy and Germination of The Baker’s Yeast S. cerevisiae Spore Ivan Pirkov Göteborg University

2. Aim of This Project The aim of my project is to uncover how eukaryotic cells maintain dormant stages and how they are again reactivated We are using baker’s yeast Saccharomyces cerevisiae as model organism S. cerevisiae produces a dormant stage, the yeast spore

3. Spore Dormancy Spores are not completely inactive –Have approx. 5% activity compared to vegetative cells –Both transcription and translation is taking place mRNA is associated mainly with ribosomes and is capped Brengues et al (2002), JBC, 277:40505-40512

4. Spore Germination RNA and protein synthesis increase within minutes upon addition of glucose to yeast spores Brengues et al (2002), JBC, 277:40505-40512 Does not require oxygen Is most efficient when a readily fermentable carbon source is present – e.g. glucose, fructose, galactose –Only carbon source is essential for germination initiation –Metabolism of the carbon source is necessary for germination, mere presence is not enough Herman and Rine (1997), EMBO J, 16:6171-6181

5. Glucose sensing of S. cerevisiae Y55 spores

6. Spore Germination Protein synthesis is needed for germination, at least during early stages Has a commitment-step –Is a transition point in germination where there is no return back to dormancy –Spores will complete germination even if the germination signal is removed Herman and Rine (1997), EMBO J, 16:6171-6181

7. Commitment-step of S. cerevisiae Y55 spores

8. On-going projects Long-term experiment on spore dormancy Microarray on vegetative cells vs. spores

9. Long-term experiment on spore dormancy When can it be considered that a spore has truly entered dormancy? –As soon as it has developed, 1 week after, 1 month etc. No easy answer Many mRNAs are abundant in the spore after the sporulation process, but decrease over time –Do the mRNA levels stabilize during longer dormancy –How will it affect the microarray experimental results –Which spores are suitable to use as control in microarray

10. StartMonth 2Month 4 4C4C30  C4C4C4C4C4C4C4C4C4C4C R1R2 R1 R2R1R2 Long-term experiment on spore dormancy Northern blots will be run on these sample with suitable mRNAs as targets

11. Evaluate which genes are up or down regulated in the resting spore compared to the exponentially growing vegetative cell Problems –Too high background –mRNA from spores seems more difficult to label  gives weaker signals compared to mRNA from vegetative cells Some information can be extracted –mRNAs that seem to be up-regulated (and retained) in the spore compared to the vegetative cell, are those for genes expressed during sporulation, late stationary phase, respiratory growth, stress responses –mRNAs which are needed for growth, e.g. ribosomal proteins, are down regulated in the spore compared to the vegetative cell Microarray on vegetative cells vs. spores

12. The main project in the near future The focus will be on microarray on germinating spores to find out which genes and pathways are involved in the germination process –Samples will be taken during the first two hrs of germination upon addition of growth medium Problems to be solved –Synchronized spores are preferred to get good resolution –Attempts have been made to synchronize the spores but with moderate success