Global Transcriptional Responses of Fission Yeast
to Glucose Starvation Stress

Technische Universität Bergakademie Freiberg
Fakultät für Chemie und Physik
Department of Molecular Genetics and
Microbiology, State University of New York, Stony Brook, New York

Michael Sassen

2005

TABLE OF CONTENT

1. Introduction ...  3
1.1 Schizosaccharomyces pombe as a Model System  ...   3
1.2 Why Investigating Signaling Cascades?  ...  . 4
1.3 The SAPK Pathway  ...   5
1.4 The cAMP Pathway ...  9
1.5 The Pheromone Pathway ...  12
1.6 Downstream the PKA and SAPK pathways  ...   15
1.7 Transporters in Fission and Budding Yeast ...  15
1.8 Glycolysis, Gluconeogenesis and Glycerol Metabolism  ...   18
1.9 Microarrays  ...   19
1.10 Thesis Goal  ...  24

2. Materials ...  25
2.1 Sources of Used Chemicals, Enzymes, and Kits ...   25
2.2 S. pombe Strains ...   25
2.3 Solutions and Yeast Media  ...   26
2.4 Equipment  ...   27

3. Methods  ...  28
3.1 Experimental Design  ...  28
3.2 Growth of S.pombe Strains  ...  28
3.3 Harvesting Cells ...   29
3.4 RNA Extraction ...   29
3.5 Sample Preparation  ...  30
3.6 Microarray  ...   33

4. Results ...  36
4.1 Genes Up-Regulated upon Glucose Starvation ...   37
4.2 Highly Induced Genes of the Carbohydrate Metabolism ...  41
4.3 Genes Involved in Mating/Meiosis  ...   44
4.4 Genes Involved in Global Transcriptional Regulation  ...   48
4.5 Hexose Transporters  ...  50
4.6 cAMP, SAPK, Pheromone Pathway Genes ...   53
4.7 Glucose Starvation vs. Oxidative Stress  ...   55
4.8 Glucose starvation vs. Nitrogen starvation ...  57
4.9 Down-Regulated Processes  ...   57
4.10 How does Gene Expression Change in a spc1 Deletion Mutant?  ...  59
4.11 PombePerl  ...   61

5. Discussion  ...  63
5.1 Changes in Metabolic Pathways ...  64
5.2 Stress Activated Signaling Pathways  ...   65
5.3 Changes of a Cell’s Global Processes  ...  67
5.4 Effects of a spc1 Deletion on Signaling Pathways  ...  69
5.5 Summary and Outlook ...   69

Acknowledgements ...   71

Figures and Tables  ...  72

References ...  74

Appendices  ...  81

Chapter 1

“Adversity has the effect of eliciting talents, which in
prosperous circumstances would have lain dormant.”

Horace (65 – 8 BC)

1. Introduction

1.1 Schizosaccharomyces pombe as a Model System

S. pombe functions as a suitable model system since it is easy and inexpensive to rear, has a convenient size, a short life cycle, and is genetically manipulable. As a unicellular eukaryote, the fission yeast S. pombe can exist either in a haploid or diploid state and possesses two different mating types (h+ and h-). The wild type, however, is h90, which means it can switch mating type.

Figure 1.01: Left, picture of S. pombe cells
At top are two dividing cells in late mitotic phase, showing the fission yeast typical septum at the point of cytoplasmic division. The lower cell is in early M phase, having its chromosomes already segregated.

Figure 1.02: Right, fission yeast cell cycle
Diagrammatic representation of the S. pombe cell cycles with the interchange between the two occurring in G1 phase (Figure obtained and used with permission from Trevor Pemberton, University of Sussex).

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S. pombe can undergo two different life cycles, either the vegetative (mitotic) cycle or the sporulation (meiotic) cycle, depending on the environment it is living in. These two cycles are shown in figure 2 with the change between the two occurring in cells at the G1 stage of the mitotic cycle. Under laboratory conditions, given all nutrients required, S. pombe prefers the haploid state. This makes it a favorable organism for genetic research since it ensures that introduced mutations are not masked by another wild type allele.

There are many similarities between relevant physiological processes in yeast and mammalian cells as supported by the cloning of the human homologs of yeast genes. Some physiological processes (e.g., mitosis, cell division) of S. pombe are more similar to those of human cells than those of the budding yeast Saccharomyces cerevisiae. Like human cells, S. pombe has a distinct G2 phase so a major checkpoint control is the decision to go from G2 to M (Russell and Nurse, 1986).

Assignment of distinct genes to different pathways has been obtained through epistasis analysis in which the phenotype of a double mutant strain is compared to the corresponding single mutant strain. Moreover, the genomes of S. cerevisiae and S. pombe were sequenced by an international group of laboratories. In addition, the availability of genome databases describing genes and predicted pathways of simple organisms could help in drug discovery programs. S.pombe is already used in a variety of applied researches, such as cancer and AIDS (Acquired Immuno Deficiency Syndrome) research (http://www.childrensmemorial.org/).

1.2 Why Investigating Signaling Cascades?

Since the major signaling pathways and cellular processes involved in a cell’s response to cytotoxic agents are conserved between yeasts and mammalian cells, simple eukaryotic systems can be valuable models for the identification of cellular/molecular mechanisms of sensitivity to drugs (Perego et al., 2000). The availability of the genomic sequence of these organisms as well as of new technologies (microarrays, proteomics) is expected to allow the identification of potential drug targets - the drug discovery process is already moving toward a genomic orientation. Signaling molecules regulate a variety of biological processes including cell cycle control, development, morphogenesis, and cellular response to environmental stress. Protein kinases are major signaling molecules found in eukaryotic cells. If scanning the entire fission yeast genome one finds the presence of 106 eukaryotic protein kinase domain-containing proteins. 44% (or 80%) of these known and putative protein kinases in fission yeast have orthologs (or nearest homologs) in mammalian systems. This suggests a conserved mechanism for signal transduction. Essential for vegetative growth are about 16% out of the total 106 protein kinases. Half of the dispensable (non-essential) protein kinases are important during growth under various stress conditions (Peng et al., 2004).

Figure 1.03: Three signaling cascades 
Three pathways relay stress related signals in S .pombe cells. The pheromone and SAPK pathway are signaling cascades with a MAP kinase as central element. The cAMP pathway mediates its signal via the second messenger cAMP. All three cascades have common downstream target genes which transcription is activated or inhibited, depending on extracellular signals.

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Two well characterized signal transduction cascades which regulate virulence and fungal development are the MAP (Mitogen Activated Protein) kinase and cAMP (3’,5’- cyclic Adenosine MonoPhosphate) signaling cascades. While the processes regulated by these cascades in fungi are as diverse as the fungi themselves, the components involved in signal transduction are remarkably conserved (D′Souza and Heitman, 2001) (see figure 1.04). Therefore, learning about the mechanisms of signal transduction in S.pombe can help us to better understand and subsequently perhaps control the virulence of human pathogens such as Cryptococcus neoformans or the plant pathogen Ustilago maydis. Since it is reasoned that the cAMP and MAP kinase signaling pathways interact with each other (D′Souza and Heitman, 2001), it is important to look at the signaling network as a whole and not only investigate each pathway separately. Together with a third signaling pathway, a pheromone activated MAPK cascade, these two pathways are of central concern in this thesis.

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