Characterization of small non-coding RNAs regulating outer membrane synthesis in the model pathogen Salmonella Typhimurium:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Berlin
Mensch & Buch Verl.
2009
|
| Schriftenreihe: | Biology
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | VIII, 153 S. Ill., graph. Darst. |
| ISBN: | 9783866646674 3866646674 |
Internformat
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| 100 | 1 | |a Pfeiffer, Verena |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Characterization of small non-coding RNAs regulating outer membrane synthesis in the model pathogen Salmonella Typhimurium |c Verena Pfeiffer |
| 264 | 1 | |a Berlin |b Mensch & Buch Verl. |c 2009 | |
| 300 | |a VIII, 153 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 490 | 0 | |a Biology | |
| 502 | |a Berlin, Freie Univ., Diss., 2009 | ||
| 650 | 0 | 7 | |a Salmonella typhimurium |0 (DE-588)4121604-0 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Membranproteine |0 (DE-588)4130026-9 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Small RNA |0 (DE-588)7501213-3 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Genregulation |0 (DE-588)4122166-7 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Salmonella typhimurium |0 (DE-588)4121604-0 |D s |
| 689 | 0 | 1 | |a Membranproteine |0 (DE-588)4130026-9 |D s |
| 689 | 0 | 2 | |a Genregulation |0 (DE-588)4122166-7 |D s |
| 689 | 0 | 3 | |a Small RNA |0 (DE-588)7501213-3 |D s |
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Datensatz im Suchindex
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| adam_text | IMAGE 1
INDEX
TABLE OF CONTENTS
1 INTRODUCTION 1
1.1 DISCOVERY AND RELEVANCE OF SMALL NON-CODING RNAS IN BACTERIA 2
1.2 GENE REGULATION BY SMALL NON-CODING RNAS IN BACTERIA 3
1.2.1 TITRATION/REGULATION OF PROTEINS BY SRNAS 3
1.2.2 POSTTRANSCRIPTIONAL REPRESSION AND ACTIVATION OF GENE EXPRESSION 4
1.3 SRNAS THAT REGULATE OUTER MEMBRANE PROTEINS 5
1.3.1 BIOGENESIS AND FUNCTION OF OUTER MEMBRANE PROTEINS 6
1.3.2 REGULATION OF OUTER MEMBRANE PROTEINS BY SRNAS 7
1.4 PROTEIN FACTORS NEEDED FOR SRNA-MEDIATED REGULATION 9
1.4.1 THE RNA CHAPERONE HFQ 9
1.4.2 RIBONUCLEASES 10
1.5 THE PATHOGEN SALMONELLA ENTERICA SEROVARTYPHIMURIUM AS A MODEL
SYSTEM 12
1.5.1 WHAT MAKES IT A PATHOGEN 12
1.5.2 HORIZONTAL GENE TRANSFER 13
1.5.3 KNOWN AND PREDICTED SRNAS IN SALMONELLA 14
1.6 GOAL OF THE STUDY 15
2 RESULTS 16
2.1 INVR IS A SPI-1-ENCODED AND CONTROLLED NON-CODING RNA THAT REPRESSES
SYNTHESIS OF THE MAJOR OUTER
MEMBRANE PROTEIN 0 16
2.1.1 THE INVR GENE BELONGS TO THE SPI-1 GENOMIC ISLAND 16
2.1.2 INVR IS AN ABUNDANT, STABLE AND INDEPENDENT RNA 17
2.1.3 HFQ IS NEEDED FOR STABILITY OF INVR 19
2.1.4 THE INVR GENE BELONGS TO THE SPI-1 REGULON AND IS ACTIVATED BY
HILD 21
2.1.5 INVR IS NOT ENGAGED IN SPI-1 REGULATION 23
2.1.6 INVFT REPRESSES THE ABUNDANT OUTER MEMBRANE PROTEIN D 28
I
BIBLIOGRAFISCHE INFORMATIONEN HTTP://D-NB.INFO/998090034
DIGITALISIERT DURCH
IMAGE 2
INDEX
2.2 MICC, A SECOND SRNA THAT REPRESSES OMPD BY CODING SEQUENCE TARGETING
46
2.2.1 MICC IS A CONSERVED HFQ-DEPENDENT SRNA THAT IS EXPRESSED IN
SALMONELLA 46
2.2.2 MICC REPRESSES OMPC AND OMPD SYNTHESIS IN SALMONELLA
POSTTRANSCRIPTIONALLY 48
2.2.3 THE MICC BINDING SITE IS DOWNSTREAM OF THE TRANSLATIONAL START IN
THE CODING REGION OF OMPD 52
2.2.4 A12 BP ANTISENSE DUPLEX IS SUFFICIENT FOR OMPD REPRESSION IN VIVO
56
2.2.5 MICC-MEDIATED REPRESSION IS INDEPENDENT OF THE TRANSLOCATION
CAPABILITY OF OMPD 57
2.2.6 TRANSLATION OF OMPD MRNA IS NOT INHIBITED BY MICC 58
2.2.7 MICC INDUCES OMPD MRNA DECAY 60
2.2.8 MICC-INDUCED OMPD MRNA DECAY REQUIRES RNASE E 62
2.2.9 IN VITRO CLEAVAGE ASSAYS SUPPORT THE RNASE E-DEPENDENCE OF THE
MICC-MEDIATED OMPD MRNA
DECAY 63
2.2.10 TWO ADDITIONAL EXAMPLES FOR SRNA-MRNA DUPLEXES THAT MEDIATE
PROCESSING OF THE TARGET MRNA IN
THE CDS IN VIVO 69
3 DISCUSSION 71
3.1 INVR, A SPI-1-ENCODED AND REGULATED SRNA, REPRESSES SYNTHESIS OF THE
CORE GENOME-ENCODED OUTER
MEMBRANE PROTEIN D 71
3.1.1 INVR BELONGS TO THE SPI-1 REGULON 71
3.1.2 SPI-1-ENCODED INVR REGULATES THE CORE GENOME-ENCODED OMPD 72
3.1.3 INVR REPRESSES OMPD DIFFERENTLY FROM THE CANONICAL MECHANISM OF
SRNA-MEDIATED REGULATION 74
3.1.4 MODEL FOR THE REGULATION OF CORE GENOME-ENCODED OMPD BY THE SPI-1
EXPRESSED SRNA INVR 76
3.2 MICC, A SECOND SRNA THAT REPRESSES SYNTHESIS OF OMPD BY TARGETING OF
THE CDS 77
3.2.1 A NOVEL TYPE OF SRNA-MEDIATED REPRESSION: CDS AS TARGET FOR SRNAS
77
3.2.2 AN RNASE E-DEPENDENT ENDORIBONUDEOLYTIC DECAY PATHWAY IS CRUCIAL
FOR REPRESSION OF OMPD IN THE
CDS 78
3.2.3 INVOLVEMENT OF THE C-TERMINUS OF RNASE E IN OMPD MRNA REPRESSION
81
3.2.4 MODE OF ACTION OF CDS TARGETING SRN AS 82
3.3 AN INCREASING SRNA POOL REGULATES THE OMP NETWORK 83
4 MATERIAL 85
4.1 MATERIAL 85
4.2 MEDIA 92
4.2.1 BACTERIAL CELL CULTURE 92
N
IMAGE 3
INDEX
4.2.2 EUKARYOTIC CELL CULTURE 94
4.3 BACTERIAL STRAINS AND PLASMIDS 94
5 METHODS 103
5.1 STERILIZATION 103
5.2 BACTERIAL CELL CULTURE 103
5.2.1 GROWTH CONDITIONS 103
5.2.2 PREPARATION OF ELECTROCOMPETENT SALMONELLA CELLS 104
5.2.3 TRANSFORMATION OF CHEMICALLY COMPETENT E. COLI CELLS 104
5.3 MUTANT CONSTRUCTION IN SALMONELLA TYPHIMURIUM 104
5.3.1 ONE-STEP INACTIVATION OF CHROMOSOMAL GENES 104
5.3.2 RESISTANCE REMOVAL FOLLOWING CHROMOSOMAL ONE-STEP TNACTIVATION 105
5.3.3 P22 TRANSDUCTION 105
SA EUKARYOTIC CELL CULTURE 106
5.4.1 HELA CELL ADHESION AND INVASION (GERITAMI* PROTECTION) ASSAY 106
5.4.2 11-8 ELISA 106
5.4.3 MACROPHAGE SURVIVAL ASSAY 107
5.4.4 CYTOTOXICITY ASSAY 107
5.4.5 TEM-1 SS-LACTAMASE ASSAY 107
5.5 ANIMAL INFECTIONS 108
5.5.1 PERORAL AND INTRAPERITONEAL INFECTIONS OF BALB/C MICE 108
5.5.2 ORAL INFECTIONS USING THE SALMONELLA TYPHIMURIUM COLITIS MODEL 108
5.6 NUCLEIC ACIDS TECHNIQUES 110
5.6.1 NUCLEIC ACIDS GEL ELECTROPHORESES 110
5.7 RNA TECHNIQUES 112
5.7.1 RNA ISOLATION 112
5.7.2 IN VITRO TRANSCRIPTION, BACKBONE-LABELING AND 5 END-LABELING OF
RNA 113
5.7.3 GENERATION OF RADIOLABELED PROBES FOR RNA DETECTION 114
5.7.4 NORTHERN BLOT 115
5.7.5 STABILITY EXPERIMENTS 116
5.7.6 DETERMINATION OF IN VIVO ABUNDANCE OF INVR 116
5.7.7 TRANSCRIPTOMIC EXPERIMENTS 116
IN
IMAGE 4
INDEX
5.7.8 QUANTITATIVE RT-PCR (QRT-PCR) 116
5.7.9 ELECTROPHORETIC MOBILITY SHIFT ASSAYS (EMSA) 117
5.7.10 IN VITRO STRUCTURE PROBING (RNA FOOTPRINTING) 118
5.7.11 PRIMER EXTENSION ANALYSIS 119
5.7.12 RAPID AMPLIFICATION OF CDNA ENDS (5 RACE) 120
5.7.13 RAPID AMPLIFICATION OF CDNAENDS (3 RACE) 120
5.7.14 30S RIBOSOME TOEPRINTS 121
5.7.15 IN VITRO RNASE E CLEAVAGE ASSAY 122
5.8 PROTEIN TECHNIQUES 123
5.8.1 PREPARATION OF WHOLE CELL PROTEIN FRACTION 123
5.8.2 PREPARATION OF MEMBRANE FRACTIONS 123
5.8.3 PREPARATION OF SECRETED PROTEIN FRACTION 123
5.8.4 ONE-DIMENSIONAL SDS PAGE 124
5.8.5 PROTEIN QUANTIFICATION BY FLUORESCENT STAIN 124
5.8.6 WESTERN BLOT 125
5.8.7 GFP REPORTER ASSAYS (FLUORESCENCE MEASUREMENTS) 125
5.8.8 IN VITRO TRANSLATION ASSAY USING RECONSTITUTED 70S RIBOSOMES 125
SUMMARY 127
ZUSAMMENFASSUNG 129
OUTLOOK 131
ACKNOWLEDGEMENTS 132
PUBLICATIONS 133
ABBREVIATION INDEX 136
REFERENCES 140
RV
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| author_facet | Pfeiffer, Verena |
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| dewey-search | 572.6964529344 |
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| dewey-tens | 570 - Biology |
| discipline | Biologie |
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| id | DE-604.BV026724970 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T23:17:52Z |
| institution | BVB |
| isbn | 9783866646674 3866646674 |
| language | English |
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| physical | VIII, 153 S. Ill., graph. Darst. |
| publishDate | 2009 |
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| spelling | Pfeiffer, Verena Verfasser aut Characterization of small non-coding RNAs regulating outer membrane synthesis in the model pathogen Salmonella Typhimurium Verena Pfeiffer Berlin Mensch & Buch Verl. 2009 VIII, 153 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Biology Berlin, Freie Univ., Diss., 2009 Salmonella typhimurium (DE-588)4121604-0 gnd rswk-swf Membranproteine (DE-588)4130026-9 gnd rswk-swf Small RNA (DE-588)7501213-3 gnd rswk-swf Genregulation (DE-588)4122166-7 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Salmonella typhimurium (DE-588)4121604-0 s Membranproteine (DE-588)4130026-9 s Genregulation (DE-588)4122166-7 s Small RNA (DE-588)7501213-3 s DE-188 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=022265433&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Pfeiffer, Verena Characterization of small non-coding RNAs regulating outer membrane synthesis in the model pathogen Salmonella Typhimurium Salmonella typhimurium (DE-588)4121604-0 gnd Membranproteine (DE-588)4130026-9 gnd Small RNA (DE-588)7501213-3 gnd Genregulation (DE-588)4122166-7 gnd |
| subject_GND | (DE-588)4121604-0 (DE-588)4130026-9 (DE-588)7501213-3 (DE-588)4122166-7 (DE-588)4113937-9 |
| title | Characterization of small non-coding RNAs regulating outer membrane synthesis in the model pathogen Salmonella Typhimurium |
| title_auth | Characterization of small non-coding RNAs regulating outer membrane synthesis in the model pathogen Salmonella Typhimurium |
| title_exact_search | Characterization of small non-coding RNAs regulating outer membrane synthesis in the model pathogen Salmonella Typhimurium |
| title_full | Characterization of small non-coding RNAs regulating outer membrane synthesis in the model pathogen Salmonella Typhimurium Verena Pfeiffer |
| title_fullStr | Characterization of small non-coding RNAs regulating outer membrane synthesis in the model pathogen Salmonella Typhimurium Verena Pfeiffer |
| title_full_unstemmed | Characterization of small non-coding RNAs regulating outer membrane synthesis in the model pathogen Salmonella Typhimurium Verena Pfeiffer |
| title_short | Characterization of small non-coding RNAs regulating outer membrane synthesis in the model pathogen Salmonella Typhimurium |
| title_sort | characterization of small non coding rnas regulating outer membrane synthesis in the model pathogen salmonella typhimurium |
| topic | Salmonella typhimurium (DE-588)4121604-0 gnd Membranproteine (DE-588)4130026-9 gnd Small RNA (DE-588)7501213-3 gnd Genregulation (DE-588)4122166-7 gnd |
| topic_facet | Salmonella typhimurium Membranproteine Small RNA Genregulation Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=022265433&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT pfeifferverena characterizationofsmallnoncodingrnasregulatingoutermembranesynthesisinthemodelpathogensalmonellatyphimurium |