Mechanisms of Dormancy and Mechanisms of Dormancy and Germination of The Baker’s Germination of The Baker’s Yeast Yeast S. cerevisiaeS. cerevisiae Spore Spore

Ivan PirkovIvan PirkovGöteborg UniversityGöteborg University

Aim of This ProjectAim 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

Spore DormancySpore 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

Spore GerminationSpore 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

Glucose sensing of Glucose sensing of S. cerevisiaeS. cerevisiae Y55 spores Y55 spores

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Spore GerminationSpore 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

Commitment-step of Commitment-step of S. cerevisiaeS. cerevisiae Y55 spores Y55 spores

Commitment-step of S. cerevisae Y55 spores

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On-going projectsOn-going projects

• Long-term experiment on spore dormancy

• Microarray on vegetative cells vs. spores

Long-term experiment on spore dormancyLong-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

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4C 30C 4C4C4C 4C4C 30C30C 30C30C 30C

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Long-term experiment on spore dormancyLong-term experiment on spore dormancy

• Northern blots will be run on these sample with suitable mRNAs as targets

• 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. sporesMicroarray on vegetative cells vs. spores

The main project in the near futureThe 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