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

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RESULTS Set A Set B CSH50 CSH50mut Exp.1 Exp.2 ME ME ME ME ME ME TS TS LZ41 LZ54 LZ41 LZ41mut LS LS LZ41mut LZ54mut LZ54 LZ54mut (compared to mutant LZ54) are those that are associated with DNA relaxation. Similarly genes upregulated in LZ54 and mutant LZ54 background are those that are associated with hypernegatively supercoiled DNA. In the experiment 2 of set B, the wild-type transcript profile of LZ41 and LZ54 was compared to their respective mutant (either fis or hns). In this comparison the upregulated genes in wild-type LZ41 background are those that are activated by DNA relaxation in the presence of FIS/H- NS. And the upregulated genes in LZ41 mutant are those that are otherwise repressed by FIS/H-NS under relaxed DNA conditions. Similarly, comparisons of LZ54 with mutants identified genes that are activated and repressed under hypernegative DNA supercoiling conditions. The differentially expressed genes derived from the microarray experiments are those that appear consistently in at least two biological replicates and three technical replicates of the microarray experiment (see section 2.2.12 for details of biological & technical replicates). Growth phaseregulated genes Rel Hyp Act Rep Figure 11: Design of DNA microarray experiments. Set A defines the regulated genes during three phases of growth. ME, TS and LS stand for midexponential, transition to stationary and late stationary phase of growth respectively. From experiment 1 of set B, the genes associated with relaxation (Rel) and hypernegative (Hyp) supercoiling were derived, while experiment 2 was used to derive genes either activated (Act) or repressed (Rep) by FIS & H-NS under both relaxation (LZ41) and hypernegative superhelicities (LZ54). The novelty of this experimental design is in the identification of growth phase regulated genes (from set A) and simultaneous derivation of supercoiling sensitivity of 37
RESULTS these identified genes (by mapping the set B gene pools on set A). The repertoire of reported supercoiling associated genes [Peter et al., 2004] was increased 5 fold by including fis & hns mutants in addition to wild-type. This experimental setup also allows the estimation of the role of chromatin proteins in distributions of effective superhelicity and organization of supercoiling-sensitive transcription. 3.1.3 Changes of supercoiling during growth In parallel to microarray experiments, high-resolution agarose gel electrophoresis was used to keep track of supercoiling changes during different growth phases of CSH50 strains and also after norfloxacin treatment in LZ strains. The global supercoiling level of DNA was determined using pUC18 reporter plasmid (Figure 12) by band counting method as previously described [Keller et al., 1975]. The mean σ values (+/- 15%) were calculated using a range of chloroquine concentrations from 0.5 to 20 μg/ml in 1% agarose gels. In the LZ strains, the relaxed topoisomers of the samples isolated from LZ41 and its derivatives demonstrated a complex pattern making it difficult to define precisely the superhelical density. During growth cycle the plasmids showed a decrease in negative supercoiling with time, consistent with the previous finding [Balke and Gralla, 1987]. The reason for such a decrease in supercoiling is attributed to the decrease in [ATP/ADP] ratio which influences the gyrase activity directly. Also consistent with the earlier reports is the increased supercoiling in hns mutant [Mojica et al., 1997] during mid-exponential phase compared to the wild-type CSH50. 38
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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 and 52: RESULTS 3 Results Growth phase-depe
Page 53: RESULTS Neither the fis nor the hns
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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