Mechanisms of Dormancy and Germination of The Bakers Yeast S' cerevisiae Spore

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Mechanisms of Dormancy and Germination of The
Bakers Yeast S. cerevisiae Spore
Ivan Pirkov Göteborg University
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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 bakers yeast Saccharomyces
  cerevisiae as model organism
• S. cerevisiae produces a dormant stage, the yeast
  spore

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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, 27740505-40512

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Spore Germination

• RNA and protein synthesis increase within minutes
  upon addition of glucose to yeast spores
• Brengues et al (2002), JBC, 27740505-40512
• 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, 166171-6181

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Glucose sensing of S. cerevisiae Y55 spores
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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, 166171-6181

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Commitment-step of S. cerevisiae Y55 spores
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On-going projects

• Long-term experiment on spore dormancy
• Microarray on vegetative cells vs. spores

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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

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Long-term experiment on spore dormancy
Start
Month 2
Month 4
4?C
30?C
4?C
4?C
4?C
4?C
4?C
30?C
30?C
30?C
30?C
30?C
R1
R2
R1
R2
R1
R2

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

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Microarray on vegetative cells vs. spores

• 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

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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

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