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Roles of glutathione in differentiation of dictyostelium discoideum : Dictyostelium discoideum의 분화에서 glutathione의 역할

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dc.contributor.advisor강사욱-
dc.contributor.author서지희-
dc.date.accessioned2017-07-14T00:47:03Z-
dc.date.available2017-07-14T00:47:03Z-
dc.date.issued2014-02-
dc.identifier.other000000016934-
dc.identifier.urihttps://hdl.handle.net/10371/121378-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2014. 2. 강사욱.-
dc.description.abstractReduced glutathione (GSH, γ-L-glutamyl-L-cysteinylglycine) is a ubiquitous tripeptide found in almost all organisms and the most abundant non-protein thiol-containing compound in eukaryotic cells. It is known to participate in diverse cellular functions, such as antioxidant defenses, the regulation of intracellular redox status, signal transduction, cell proliferation and death, and immune responses. GSH also participates in regulation of organ differentiation.
Previously, it was reported that GSH serves important roles in normal growth and differentiation of Dictyostelium discoideum. The developmental morphology of gcsA¯ cells was dependent on the concentration of GSH which was added to culture media. In this work, to find out the precise roles of GSH during development, intracellular GSH was completely depleted and then developmental morphology was observed. Absence of GSH caused defects in the formation of multicellular aggregates. gcsA¯ cells were in a state of single cells if GSH was not supplemented. This developmental defect of gcsA¯ cells was rescued by adding exogenous GSH, γ-GC, or GSSG. But other thiol-compounds or antioxidant molecules, such as DTT, NAC, and ascorbic acid, did not compensate GSH. These results indicate that GSH itself plays essential roles rather than as an antioxidant molecule in regulating the development of Dictyostelium.
To gain more information on the developmental defect of gcsA¯ cells, the expression patterns of genes that were required to initiate development were examined. GSH-depleted gcsA¯ cells failed to decrease the expression of a growth-stage-specific gene (cprD) and failed to induce the expression of genes that encode proteins required for early development (discoidin, dscA
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dc.description.abstractdifferentiation associated protein, dia2-
dc.description.abstractcAMP receptor, carA/cAR1-
dc.description.abstractadenylyl cyclase, acaA/ACA-
dc.description.abstractand the catalytic subunit of protein kinase A, pkaC/PKA-C). Decreased expression of carA and acaA was remarkable in gcsA¯ cells. However, the developmental defect of gcsA¯ cells was not restored by cAMP stimulation or by cAR1 expression. Though constitutively expressed cAR1 induced the expression of acaA and Gα2 gene, gcsA¯ cells did not develop without GSH. These results suggest that GSH seems to work at higher step to the cAMP signaling pathway to regulate development of Dictyostelium.
YakA signaling is known the earliest response to environmental signal to initiate development and functions prior to cAMP signaling. The expression of yakA is responsible to induce the expression of differentiation-associated genes and to inhibit the expression of growth-phase genes for the initiation of development. The expression of yakA was regulated by intracellular GSH in both KAx3 and gcsA¯ cells. GSH-depleted gcsA¯ cells showed undetectably low yakA expression levels, but the expression was induced by adding GSH. The expression of yakA was in proportion to the concentration of exogenously added GSH in KAx3 cell. Further, induced yakA expression promoted the formation of multicellular aggregate in both KAx3 and gcsA¯ cells. Intracellular GSH also influenced on the expression of pufA and the activity of PKA, which are components of downstream regulators in the YakA signaling pathway. gcsA¯ cells showed increased pufA expression and lowered PKA activity compared to KAx3 cells. However, the expression of pufA and the activity of PKA were recovered to the similar level of KAx3 cells by adding GSH. Interestingly, yakA¯ cells showed similar gene expression pattern and developmental morphology to gcsA¯ cells. yakA¯ cells did not develop. The expression of carA and acaA was significantly decreased and the activity of PKA was not detected in yakA¯ cells. Exogenous GSH did not rescue the developmental defects of yakA¯ cells, but constitutively expressed YakA in gcsA¯ cells (YakAOE/gcsA¯) rescued the developmental defects of gcsA¯ cells without the addition of GSH
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dc.description.abstractYakAOE/gcsA¯ cells formed multicellular aggregates and carA and acaA were expressed without GSH. These results indicate that intracellular GSH plays indispensable roles during development by regulating the expression of yakA in Dictyostelium.
To investigate the relation between YakA and GSH further, the concentration of intracellular GSH the expression of gcsA were monitored in yakA¯ and YakAOE/KAx3 cells. yakA¯ cells showed decreased intracellular GSH levels around 40% compared to KAx3 and considerably increased gcsA expression. However, constitutive expression of YakA in KAx3 cells (YakAOE/KAx3 cells) did not significantly influence on the intracellular GSH level and gcsA expression, indicating that GSH regulates the expression of yakA but YakA did not regulate intracellular GSH. Decreased intracellular GSH concentration might be caused by hypersensitiveness to oxidative stress of yakA¯ cells and leads to accumulation of gcsA transcripts by the feedback regulation of GSH.
Taken together, these findings suggest that GSH plays an essential role in the transition from growth to differentiation by modulating the expression of the genes encoding YakA as well as components that act downstream in the YakA signaling pathway in Dictyostelium.
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dc.description.tableofcontentsABSTRACT i
CONTENTS iv
LIST OF FIGURES vii
LIST OF TABLES x
LIST OF ABBREVIATIONS xi

I. INTRODUCTION 1
1. Glutathione 2
1.1. An overview 2
1.2. The enzymatic synthesis of glutathione 4
1.3. The roles of glutathione in cellular reactions 6
1.4. The roles of glutathione in development 9
2. Dictyostelium discoideum 11
2.1. Properties as a model organism 11
2.2. The transition from growth to development 13
2.3. Intracellular signals required for the initiation of development 16
2.3.1. Prestarvation factors 17
2.3.2. Conditioned medium factors 18
2.4. The early events induced by starvation 19
2.4.1. The cAMP signaling pathway 19
2.4.2. The YakA signaling pathway 26
3. Aims of this study 29

II. MATERIALS AND METHODS 31
1. Strains and culture conditions 32
1.1. Dictyostelium strains and culture conditions 32
1.1.1. Dictyostelium strains 32
1.1.2. Culture conditions 32
1.2. Bacterial strains and culture conditions 33
1.2.1. Escherichia coli strains for gene cloning 33
1.2.2. Klebsiella pneumoniae strain as a food source of Dictyostelium 33
2. Depletion of GSH 33
3. Development of Dictyostelium discoideum 35
3.1. Development on non-nutrient agar plates 35
3.2. Development in non-nutrient buffer 35
4. Transformation of Dictyostelium discoideum 37
5. Genetic manipulation methods 39
5.1 Isolation and subcloning of carA from Dictyostelium discoideum 39
5.2 Isolation and subcloning of yakA from Dictyostelium discoideum 41
5.3. Polymerase chain reaction (PCR) 41
5.4 Real-time reverse transcriptase-polymerase chain reaction (Real-time RT-PCR) 42
5.5 Total RNA extraction and Northern blotting analysis 43
6. Measurement of PKA activity 43
7. Measurement of glutathione concentration 45

III. RESULTS 47
1. The roles of GSH in development of Dictyostelium discoideum 48
1.1. Complete depletion of GSH in Dictyostelium 48
1.2. The roles of GSH in development on agar plates 50
1.3. The roles of GSH in aggregation processes 50
1.4. Irreplaceable role of GSH by antioxidant molecules 54
2. Developmental properties of the GSH-depleted gcsA¯ cells 58
3. The roles of GSH in the regulation of cAMP signaling 59
3.1. The expression of genes related with the cAMP signaling system in gcsA¯ cells 59
3.2. The effect of cAMP stimulation on development of gcsA¯ cells 62
3.3. The effect of cAR1 expression on development of gcsA¯ cells 63
4. The role of GSH in the regulation of YakA signaling 69
4.1. The expression of yakA in gcsA¯ cells 69
4.2. The effect of intracellular GSH on the expression of yakA 71
4.3. The expression of YakA downstream regulators in gcsA¯ cells 76
4.3.1. The expression of pufA 79
4.3.2. The gene expression and the enzymatic activity of PKA 80
5. Developmental properties of yakA¯ cells 80
5.1. The developmental morphology of yakA¯ cells 80
5.2. The expression of developmental genes in yakA¯ cells 83
5.3. The effect of GSH on the developmental morphology of yakA¯ cells 83
6. The role of GSH in the regulation of YakA signaling 87
6.1. The effect of YakA expression on the developmental morphology of gcsA¯ cells 87
6.2. The effect of YakA expression on the expression of early developmental genes in gcsA¯ cells 91
6.3. The effect of YakA expression on the concentration of intracellular GSH 91
7. Relation between YakA and intracellular GSH 98
7.1. The intracellular contents of GSH in yakA¯ cells 98
7.2. The expression of gcsA in yakA¯ cells 99

IV. DISCUSSION 103
V. REFERENCES 113
국문초록 133
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dc.formatapplication/pdf-
dc.format.extent11520155 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectGlutathione-
dc.subjectYakA-
dc.subjectTransition from growth to differentiation-
dc.subjectDictyostelium discoideum-
dc.subject.ddc570-
dc.titleRoles of glutathione in differentiation of dictyostelium discoideum-
dc.title.alternativeDictyostelium discoideum의 분화에서 glutathione의 역할-
dc.typeThesis-
dc.contributor.AlternativeAuthorJi-Hui Seo-
dc.description.degreeDoctor-
dc.citation.pagesxi, 135-
dc.contributor.affiliation자연과학대학 생명과학부-
dc.date.awarded2014-02-
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