Functional analysis of MDY2, a novel gene required for mating of the yeast Saccharomyces cerevisiae [Elektronische Ressource] / vorgelegt von Zheng Hu

Ag Hollenberg

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2004
• Nombre de lectures: 29
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

Functional Analysis of MDY2, a Novel Gene Required for
Mating of the Yeast Saccharomyces cerevisiae




INAUGURAL-DISSERTATION

zur

Erlangung des Doktorgrades

der

Mathematisch-Naturwissenschaftlichen Fakultät

der Heinrich-Heine-Universität Düsseldorf





vorgelegt von

Zheng HU

aus Hangzhou, China




Düsseldorf 2004






Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf











Referent: Prof. Dr. C. P. Hollenberg

Korreferent: Prof. Dr. Th. Lisowsky

Korreferent: Priv.-Doz. Dr. M. Ramezani Rad




Tag der mündlichen Prüfung: 25. Mai 2004



Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes
Contents 1
Contents


1. Introduction 5
1.1 Mating process of S. cerevisiae 5
1.2 Pheromone response pathway of S. cerevisiae 7
1.3 The Ste5 scaffold 9
1.4 The MAPK Fus3 11
1.5 The aim of this study 14

2 Materials and Methods 15
2.1 Materials 15
2.1.1 Yeast strains and media 1
2.1.2 Escherichia coli strains and media 20
2.1.3 Plasmids and oligonucleotides 20
2.2 Chemicals, enzymes and antibodies 24
2.2.1 Chemicals 24
2.2.2 Enzymes 25
2.2.3 Antibodies 25
2.3 Methods 25
2.3.1 Mating assay 25
2.3.2 Isolation and characterization of Plasmid DNA 27
2.3.3 Transformation of S. cerevisiae 28
2.3.4 Transformation in E. coli 29
2.3.5 Manipulation of DNA 29
2.3.6 Polymerase Chain Reaction (PCR) 30
2.3.7 Determination of pheromone production (halo assay) 31
2.3.8 Pheromone response assay (halo assay) 31
2.3.9 Determination of β-galactosidase activity on plates 32
2.3.10 Determination of β-galacctivity in liquid phase 32
2.3.11 Determination of shmoo and budding indexes 33
2.3.12 Microscopy 33
2.3.13 Gene disruption and verification of disruption by PCR 34
2.3.14 Two-hybrid assay 34
2.3.15 SDS-Polyacrylamide Gel Electrophoresis of proteins 35
Contents 2
2.3.16 Immunolabelling of protein blots 36
2.3.17 Protein interaction analysis 37
2.3.18 Galactose depletion assay 38
2.3.19 Invasive growth assay 38
2.3.20 Analysis of sensitivity to hyperosmolarity 39
2.3.21 Bio-informatics 39

3 Results 40
3.1 Functional analysis to specify the reduced mating effciency of single gene deletion mutants of
S. cerevisiae 40
3.1.1 Assessment of the mating efficiency of the 64 single gene deletion mutants 4
3.1.1.1 Semi-quantitative mating analysis 40
3.1.1.2 Complementation of the mating defects 43
3.1.1.3 Data analysis and selection of candidates for further study 43
3.1.1.4 Quantitative mating analysis of 15 mutants 46
3.1.2 Pheromone production by the 15 mutants 47
3.1.2.1 α-factor production by the 15 mutants 47
3.1.2.2 a-factor production by the 15 mutants 49
3.1.3 Analysis of the pheromone response in the 15 mutants 49
3.2 Characterization of the mating defective mutants with respect to morphogenesis, cell fusion
and nuclear fusion 51
3.2.1 Analysis of mutants for the ability to activate the pheromone response pathway 51
3.2.1.1 Activation of the report gene FUS1::lacZ 51
3.2.1.2 Shmoo index 54
3.2.2 The kinetics of zygote formation 54
3.2.3 Analysis of the 15 mutants by fluorescence microscopy for defects in cell fusion and nuclear
fusion 56
3.3 Functional analysis of the role of MDY2 in mating of S. cerevisiae 58
3.3.1 Disruption of MDY2 and phenotypic analysis of the mdy2 deletant 58
3.3.1.1 Disruption of MDY2 in S.cerevisiae strain W303 58
3.3.1.2 Deletion of MDY2 results in reduced mating efficiency 59
3.3.1.3 Analysis of the shmoo and budding indexes of mdy2 deletion mutants 60
3.3.1.4 α-Pheromone production is reduced in MAT α mdy2 deletion strains 61
3.3.1.5 Impaired growth arrest upon exposure of mdy2 mutant cells to α-factor 62
Contents 3
3.3.1.6 Analysis of pheromone induced Fus1-lacZ reporter activity in mdy2 cells 63
3.3.2 Analysis of the effects of MDY2 overexpression 63
3.3.2.1 Overexpression of MDY2 increases mating efficiency 63
3.3.2.2 Increased dosage of MDY2 in the mdy2 mutants restores the wild-type rate of shmoo formation 64
3.3.2.3 Overexpression of MDY2 corrects the defect in α-pheromone production observed in the mdy2
mutan 65
3.3.2.4 MDY2 in high copy enhances pheromone induced G arrest of the mdy2 mutant 6 1
3.3.2.5 Overexpression of MDY2 leads to an increase in pheromone induced FUS1-lacZ expression 66
3.3.3 Identification of Mdy2 interaction partners 66
3.3.2.1 Two-hybrid analysis 66
3.3.2.2 Pull down analysis 68
3.3.4 Mdy2 and the dynamics of MAPK Fus3 modification 69
3.3.5 Double deletion of MDY2 and KSS1 affects pheromone induced G arrest 70 1
3.3.6 The effect of Mdy2 on the level of the scaffold protein Ste5 71
3.3.6.1 The level of Myc-Ste5 in wild-type and mdy2 mutant strains after pheromone induction, as
revealed by the galactose depletion assay 71
3.3.6.2 The level of GST-Ste5 in wild-type and mdy2 mutant strains after pheromone induction , as
revealed by the galactose depletion assay 72
3.3.6.3 The level of the GST-Ste5 in wild-type and mdy2 mutant strains during vegetative growth, as
monitored by the galactose depletion assay 72
3.3.7 Subcellular localization of Mdy2 73
3.3.7.1 Overexpression of GFP-MDY2 73
3.3.7.2 Expression of GFP-MDY2 under its own promoter 74
3.3.8 The effect of MDY2 on the invasive growth pathway and HOG pathway 75
3.3.8.1 Invasive growth test 75
3.3.8.2 Osmosensitivity assay 75

4 Discusion 7
4.1 Identification of novel genes required for mating of S. cerevisiae 77
4.2 Characterization of 15 deletion mutants with reduced mating efficiency 80
4.3 Functional analysis of MDY2 in mating of S. cerevisiae 84
4.3.1 MDY2 is required for regulation of the mating pheromone response pathway 85
4.3.2 Mdy2 interacts with components of the mating pathway and localizes in nucleus 85
4.3.3 Mdy2 and the dynamics of MAPK Fus3 modification 87
Contents 4
5 Sumary 90
6 Refrencs 91
7 Abreviatons 10


Introduction 5
1. Introduction


One of the oldest questions in biology is how cells sense and discriminate between various
environmental stimuli and then translate these inputs into an appropriate intracellular response (review
Dohlman and Thorner, 2001). In the S. cerevisiae, environmental stimuli of various kinds can induce
a diversity of processes including mating and spore formation, as well as developmental responses
triggered by starvation, hi