Coordinated regulation of gene expression by E ... - Jacobs University

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MATERIALS AND METHODS measure of opening of the two strands of DNA during processes such as initiation of transcription. All the components (except KMnO4) are mixed on ice and equilibrated at 37oC for 5 minutes before adding 2 μl of 100 mM KMnO4. After exactly 30 seconds, the footprint is stopped by the addition of 2 μl of β-mercaptoethanol (14M). To this 100 μl of TE (pH 8.0) is added and then the DNA is extracted with phenol: chloroform and precipitated as described above. The pellet of footprinted DNA is dissolved in 15 μl of water and is subjected to primer extension with labeled primer using Taq polymerase and 1 mM final concentration of dNTP mix for six cycles in a PCR machine (Peltier Thermal Cycler PTC 100 & PTC 200 from MJ research). The product is resolved on a 6% polyacrylamide gel along with a sequencing reaction and visualized by phosphorimaging. The di-deoxynucleotide termination sequencing reaction of the plasmid DNA and the primer used for footprinting and PCR was performed using commercially available sequencing kit from USB (Sequenase Version 2.0 DNA Sequencing Kit) following the manufacturers instruction. 2.2.19 β-Galactosidase assay Beta-galactosidase assay was performed following the protocol of Sadler & Novick [Sadler & Novick, 1965]. The β-Galactosidase assay was performed on freshly diluted (1:30) overnight cultures after one hour of dilution cultures in exponential phase. β- Galactosidase activity is calculated as a function of, colorless ONPG substrate turning to yellow nitro phenol product (measured at OD420) and the time it takes to turn yellow by using the formula: β-Galactosidase activity = OD420 X 8.5 X 100 OD600 X time (minutes) 33
RESULTS 3 Results Growth phase-dependent transcription regulation has been studied in the current work at two levels of complexity, global and the local. Global regulation, explained in the first section details the effects of DNA supercoiling and chromatin proteins FIS and H-NS in coordinating the growth phase dependent transcription. In the second section the mechanism of transcriptional regulation by chromatin protein FIS and supercoiling is studied at the molecular level in a model system of a stable RNA promoter. 3.1 Growth phase dependent global transcriptional regulation Regulation of growth is managed by the interplay of chromatin proteins, topology of the DNA and the transcription machinery. Using DNA microarrays we have tried to address the relationship between the organization of chromosomal supercoiling and growth phase dependent gene expression patterns. Since chromosomal supercoiling is tightly connected to the function of abundant chromatin proteins, we used mutants of fis and hns genes known as global transcriptional regulators. We compared the expression profiles of wild-type with each mutant separately during three different phases of growth and at different superhelicities induced by addition of a topoisomerase poison. Our experimental design enabled us to reveal the relationships between the effects of chromatin proteins and supercoiling in global transcriptional regulation. 3.1.1 Growth of CSH50 and LZ strains All the strains, CSH50 wild-type, CSH50 fis::kan and CSH50 hns-205::Tn10 mutants and LZ41, LZ54 and their fis::cm and hns-205::Tn10 derivatives were grown in double YT medium. The CSH50 strains were grown for 24 hours during which three time 34
Page 1 and 2: Coordinated regulation of gene expr
Page 3 and 4: Parts of this work has been publish
Page 5 and 6: ABSTRACT promoter region determines
Page 7 and 8: ACKNOWLEDGEMENTS first impression o
Page 9 and 10: TABLE OF CONTENTS 2.2.5 Preparation
Page 11 and 12: LIST OF TABLES List of tables Table
Page 13 and 14: LIST OF FIGURES List of Figures Fig
Page 15 and 16: ABBREVIATIONS Abbreviations ∆61
Page 17 and 18: ABBREVIATIONS TBE Tris TS tyrT35U t
Page 19 and 20: INTRODUCTION Salmonella: “In more
Page 21 and 22: INTRODUCTION Figure 1: Schematic il
Page 23 and 24: INTRODUCTION UP element -35 -10 Spa
Page 25 and 26: INTRODUCTION can also influence elo
Page 27 and 28: INTRODUCTION have more base-pairs t
Page 29 and 30: INTRODUCTION Figure 6: Graphical re
Page 31 and 32: INTRODUCTION shown that H-NS reache
Page 33 and 34: INTRODUCTION formed as a repercussi
Page 35 and 36: MATERIALS AND METHODS 2 Materials a
Page 37 and 38: MATERIALS AND METHODS NaCl 10 gm YT
Page 39 and 40: MATERIALS AND METHODS LZ41 3 pyrF28
Page 41 and 42: MATERIALS AND METHODS 2.2 Methods 2
Page 43 and 44: MATERIALS AND METHODS given using a
Page 45 and 46: MATERIALS AND METHODS phenol: chlor
Page 47 and 48: MATERIALS AND METHODS Filtered (by
Page 49: MATERIALS AND METHODS free water (A
Page 53 and 54: RESULTS Neither the fis nor the hns
Page 55 and 56: RESULTS these identified genes (by
Page 57 and 58: RESULTS furthermore the colour of t
Page 59 and 60: RESULTS During the entire growth cy
Page 61 and 62: RESULTS transcripts of a growth pha
Page 63 and 64: RESULTS phosphorylation, are associ
Page 65 and 66: RESULTS Table 4: RT-PCR analysis of
Page 67 and 68: RESULTS Figure 16: Schematic repres
Page 69 and 70: RESULTS parC) which allows the supe
Page 71 and 72: RESULTS Figure 19: In vitro transcr
Page 73 and 74: RESULTS contrast the tyrTD promoter
Page 75 and 76: DISCUSSION 4 Discussion The idea of
Page 77 and 78: DISCUSSION In a study by Balke & Gr
Page 79 and 80: DISCUSSION cells maintain viability
Page 81 and 82: DISCUSSION writhe in the UAS by FIS
Page 83 and 84: DISCUSSION 4.3.2 Mechanism of trans
Page 85 and 86: CONCLUSION 5 Conclusion Regulation
Page 87 and 88: REFERENCES Bordes, P., Conter, A.,
Page 89 and 90: REFERENCES Gruber, T.M., and Gross,
Page 91 and 92: REFERENCES molecular basis for sele
Page 93 and 94: REFERENCES Pan, C.Q., Finkel, S.E.,
Page 95 and 96: REFERENCES Schroder, O., and Wagner
Page 97 and 98: REFERENCES Willenbrock, H., and Uss
Page 99 and 100: APPENDIX I gene blattner ratio p-va
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APPENDIX I gene blattner ratio p-va
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APPENDIX I Supplementary table - VI
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APPENDIX I fabB b2323 0.6336 0.0043
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APPENDIX I ibpA b3687 2.4960 0.0181
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APPENDIX I pepP b2908 1.2182 0.0368
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APPENDIX I speG b1584 0.4151 0.0001
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APPENDIX I ycjX b1321 4.0841 0.0141
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APPENDIX I yhfY b3382 1.4063 0.0453
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APPENDIX I gene blattner ratio A/B
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Statement of sources Declaration I
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