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

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MATERIALS AND METHODS ligating them in the presence of different amount of ethidium bromide. Initially the plasmid was nicked using 20 units of Nt.BstNBI, for every 20μg of DNA in a reaction volume of 50μl according to manufacturers specification The nicked plasmids (2.5μg for each reaction) were then ligated using T4 DNA ligase in the presence of different amount of ethidium bromide (concentrations ranging from 800μg/ml to 2.5μg/ml were used) in an overnight reaction. The reaction products were then extracted twice with phenol: chloroform mixture, the DNA precipitated, pelleted and re-dissolved in 10μl of TE pH 8.0. The σ values of the generated topoisomers were quantified by band counting method [Keller et al., 1975]. Briefly, mid-point of a completely relaxed plasmid (nicked and ligated without ethidium bromide) population was taken as a σ value of zero. Then by determining the mid-point of other plasmid topoisomers (ladder) and comparing it with the relaxed gives the linking number difference (∆Lk). From this the σ is calculated using the formula σ = ∆Lk / ∆Lk o , where ∆Lk o for pUC18 = 2686 bp / (10.4 bp / turn) = 258. The σ values of the plasmids from different growth phase and LZ strains were determined by comparing with the ladder. 2.2.16 In vitro transcription In vitro transcription was performed from supercoiled plasmids with distinct σ (ptyrT & ptyrTD) and linearized (∆61tyrT) plasmids. Linearized plasmids were obtained by cutting the plasmid with 10 units of BamHI for every microgram of DNA for 1 hr. The different topoisomer of ptyrT & ptyrTD were obtained by incubating 20μg of supercoiled plasmid with 25 units Vaccinia topoisomerase I in the presence of different ethidium bromide concentrations in topoisomerase buffer (10mM Tris (pH 8.0), 100mM NaCl, 20mM EDTA) for two hours at 37 o C. After obtaining the template, in vitro transcription was performed as described before [Lazarus & Travers, 1993] with 1μg of plasmid DNA, 1μl of 1 unit/μl RNAP (Bohreinger), 44nM FIS when indicated and 2.0mM NTP mix in a 50μl reaction volume. After the reaction, the RNA synthesized was extracted with phenol: chloroform and precipitated as described above. The pellet is dissolved in 10 or 20 μl of nuclease 31
MATERIALS AND METHODS free water (Ambion) for subsequent use in the primer extension reaction (10 μl for each primer). 2.2.17 Primer extension The mRNA from in vitro transcription of ∆61 tyrT constructs and from tyrT with different supercoiling was divided into two 10 μl parts to be used for primer extension with Ex103 (for tyrT transcript) and bla primers. Primer extension from total RNA from LZ strains transformed with different tyrT plasmids was performed with Ex103 and a collective primer for chromosomal rrn operon’s P1 promoter, G11, as described by Nasser et al.,[Nasser et al., 2001]. The primers (200 pmol) to be extended were radioactively labeled using 50 μci of P32 γ-ATP (Amersham Biosciences now a part of GE Healthcare) and T4 polynucleotide kinase (PNK) (Invitrogen) in a reaction volume of 20μl and the labeled primer purified using G25 spin columns (GE healthcare, formerly Amersham Biosciences). Primer extension reaction was performed from 3 μg of total RNA or mRNA from in vitro transcription both in a volume of 10 μl, 2 mM final concentration of dNTP mix, 2 pmol of labeled primer, 2 μl of 0.1M DTT, 4 μl of 5X reverse transcriptase buffer and 1 μl of LMV-reverse transcriptase. The primer extension products were separated on a 6% polyacrylamide gel and visualized by phosphorimaging (FUJI film FLA-3000 phosphorimager). 2.2.18 Potassium permanganate footprinting The components of the footprinting reaction are the same as the in vitro transcription reaction. The footprinting was carried out as described earlier [Nasser et al., 2001]. The addition of potassium permanganate (KMnO4), which oxidize preferentially the thymine nucleotide (cytosine can also be oxidized) of an open double helix, gives a 32
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: MATERIALS AND METHODS Filtered (by
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
Page 99 and 100:
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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