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

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MATERIALS AND METHODS Figure 9: Overview of comparative gene expression of wild-type & fis (example) analysis using microarrays. 2.2.13 Microarray analysis The scanned images of the spots on the arrays after quantification have to be subjected to normalization before any comparison of the gene expression is possible. Normalization of the spot intensity for a particular dye (Cy3 or Cy5) to the combined total intensity of that dye in all the spots of the array has to be performed for several reasons. It allows for comparison between microarray slides & between experiments and is necessary to eliminate the inherent difference in the intensities of Cy3 & Cy5 fluorophores. Scanned array images were analyzed using the TM4 software package from TIGR [Saeed et al., 2003]. The first step in the analysis, to quantify the spots and to do a quality check on the spots, was carried out using TIGR spotfinder software. Briefly, spot intensities were quantified and quality of each spot was verified by calculating a quality control (QC) score depending on signal-to-noise ratio of both Cy3 and Cy5 channels in the spot and shape quality of the spot. Then the p-values were calculated for each channel as result of a t-test comparing the spot pixel set and surrounding background pixel set still using the TIGR Spotfinder software. 29
MATERIALS AND METHODS Filtered (by spot intensity and spot quality) data was normalized by locally weighted linear regression [Cleveland and Devlin, 1988] and, a one-class t-test [Pan et al., 2002] was applied to the mean ratios (of wild-type to mutant or relaxed to hypernegative) from replicated experiments to obtain differentially regulated genes with significant p-values (p < 0.05) using the TIGR MIDAS software. Representation of regulated genes in the form of genome wheels was accomplished using a web-based visualization tool called the Genome viewer. The webbased program can be accessed at the web site- http://www.cmbi.ru.nl/MGV/ [Kerkhoven, 2004]. 2.2.14 Real-time PCR Each RT-PCR reaction was carried out in triplicates from the purified total RNA using QantiTect ® SYBR ® Green one-step Real-Time PCR reactions (Qiagen GmbH, Hilden, Germany) following the manual of manufacturer, using the instrument Mx3000P Real- Time cycler (Stratagene ® , La jolla, CA, USA). 2.2.15 High resolution agarose gel electrophoresis and determination of sigma (σ) Separation of the pUC18 plasmid topoisomers and their supercoiling (σ) determination, by comparing with a plasmid topoisomer ladder, was performed using high-resolution agarose gel electrophoresis. Electrophoresis was carried out in 1% agarose gel with 1X TBE buffer and chloroquine in both the gel and the buffer. Different amount of chloroquine (0.5μg/ml to 20μg/ml) was used for resolution of different topoisomers. The electrophoresis was performed at 4 o C for 24 hours at 40V. After electrophoresis, the gels were stained in ethidium bromide (0.5 μg/ml in water) and photographed. For the determination of sigma (σ), a ladder of plasmids with overlapping topoisomer distribution was generated by nicking the pUC18 plasmid DNA and then re- 30
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: MATERIALS AND METHODS phenol: chlor
Page 49 and 50: MATERIALS AND METHODS free water (A
Page 51 and 52: RESULTS 3 Results Growth phase-depe
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
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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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