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

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RESULTS points were sampled based on the optical density (OD) whereby the mid-exponential phase sample corresponds to an OD of 0.6 (Figure 10) The LZ strains which harbor drug resistant alleles of topoisomerases, gyrase (gyrA L83 ) and topoisomerase IV (parC K84 ) genes were treated with quinolone norfloxacin during mid-exponential growth before sampling. Norfloxacin used at sublethal concentration in LZ41 and LZ54 strains selectively inhibits DNA gyrase or topoisomerase IV activity thereby inducing relaxation or hypernegative supercoiling respectively. In these strains, the cells are exposed to norfloxacin for 5 minutes and RNA is then extracted [Rochman et al., 2002]. This allows the variation of transcription with overall superhelical density to be monitored from almost fully relaxed (σ = - 0.015, strain LZ41) to near physiological (σ = - 0.073, strain LZ23) to hypernegatively supercoiled (σ = - 0.095, strain LZ54) state [Zechiedrich et al., 1997]. Figure 10: Growth curves of CSH50 wild-type and the mutants. The three phases sampled for the microarray- based comparison of differential gene expression. (A) Growth curves of wild-type and fis mutant. (B) Growth curves of wild-type and hns mutant. The dots indicate the time of sampling. 35
RESULTS Neither the fis nor the hns mutations in the CSH50 or in the LZ strains affected the overall growth pattern but the growth rates of the fis and hns mutants of CSH50 were slightly different from the wild-type (data not shown). The growth rate comparisons showed differences during exponential growth stage and early stages of stationary phase. In comparison to wild-type cells the fis mutants had lower and hns mutants had higher growth rates during exponential phase. These differences are not clearly understood but correspond to the global repressing effect of H-NS and general growth-stimulatory effect of FIS (due to activation of stable RNA promoters). 3.1.2 A novel microarray based approach to derive growth phasespecific transcript profiles organized by supercoiling and chromatin proteins Two sets of microarray experiments were performed, the design of which is schematically represented in figure 11. Briefly, in set A, comparison of the transcript profiles of wild type and mutant at each growth phase allowed the identification of FIS and H-NS regulated genes during the three phases of growth (ME, TS and LS). In the second set (set B) of microarray experiments combinations of LZ strains were used to derive supercoiling associated genes (Exp.1 in Figure 11) and genes modulated by FIS and H-NS at different supercoiling (Exp.2 in Figure 11). Comparison of LZ41, LZ54 and their mutants in different combinations was performed during the mid-exponential phase, when effect of the topoisomerase inhibitory drug norfloxacin was most pervading. In set B the transcript profiles of LZ41 (norfloxacin-induced DNA relaxation), LZ54 (norfloxacin-induced hypernegative DNA) and their fis and hns mutants were compared in different combination, which is divided into experiment 1 and experiment 2 for the sake of clarity in Figure 11. Experiment 1 compares LZ41 with LZ54 and mutant (either fis or hns) of LZ41 with mutant (either fis or hns) of LZ54. The genes upregulated in LZ41 background (compared to LZ54) and mutant LZ41 background 36
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 and 50: MATERIALS AND METHODS free water (A
Page 51: RESULTS 3 Results Growth phase-depe
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
Page 101 and 102: 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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