Gene expression in plastids of higher plants [Elektronische Ressource] : evolutionary and functional aspects of different RNA polymerases ; coordinated assembly of multiprotein complexes / vorgelegt von Juliana Legen

Legen Juliana

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2002
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Gene expression in plastids of higher plants:
evolutionary and functional aspects of different RNA
polymerases – coordinated assembly of multiprotein-
complexes












Inaugural-Dissertation der Fakultät für Biologie
der Ludwig-Maximilians-Universität München











Gene expression in plastids of higher plants:
evolutionary and functional aspects of different RNA
polymerases – coordinated assembly of multiprotein-
complexes






Inaugural-Dissertation der Fakultät für Biologie
der Ludwig-Maximilians-Universität München










vorgelegt von
Juliana Legen
aus Wolfratshausen
November, 2002























1. Gutachter: Prof. Dr. Reinhold G. Herrmann
2. Gutachter: Prof. Dr. Hans Ulrich Koop


Tag der Mündlichen Prüfung: April, den 8, 2003
1. Introduction……………………………………………………………………………………1

1.1. Structure and organisation of plastid chromosomes…..………..…………………….….1
1.2. Expression of plastid chromosomes…………………………….………………………….4
1.2.1. Transcription………………………………………………………………………..5
1.2.2. Posttranscriptional RNA processing……………………………..………………6
1.2.3. Translational control of plastid gene expression……………………………..…………...9
1.3. Thylakoid membrane-located multiprotein complexes…………………………………...9
1.3.1. Synthesis and assembly of the cytochrome b /f complex………………………………10 6
1.4. Goals of this study………………………………………………………………..12

2. Materials…………………………………………………………………….…………………14

2.1. Chemicals and enzymes…………………………………………………………….……..14
2.2. Length and weight standards………………………………………...15
2.3. Antibodies…………………………………………………………………………15
2.4. Bacterial strains……………………………………………..16
2.5. Plasmids…………………………………………………………………………
2.6. Oligonucleotides…………………………………………….16
2.7. Plant growth media………………………………………………………………16
2.8. Plant material………………………………………………..17
2.9. Media for growth of bacteria…………………………………………………….17
2.10. Antibiotic stock solutions…………………………………………………………………...18
2.11. General buffers and solutions……………………………………………………………..18

3. Methods…………………………………………………………………………………...19

I. Manipulation of Nucleic Acids……………………………………………………………………..19
3.1. Isolation of nucleic acids………………………………………………………………..…..19
3.1.1. Small-scale plasmid isolation from E.coli (Miniprep)…………………...……….….……19
3.1.2. Isolation of genomic DNA……….…………………………………………………....…….19
3.1.3. Isolation of RNA……………………………………….….….19
3.1.4. Elution of nucleic acids from agarose gels…………………………………………….….20
3.1.5. Determination of nucleic acid concentrations…………………..………….…..20
3.2. Enzymatic modifications of nucleic acids……………………………………….………...20
3.2.1. Restriction analysis of DNA………………………………………………….……………..20
3.2.2. Ligation of DNA fragments………………………….…………………20
3.2.3. Dephosphorylation of plasmid DNA……………………………………………………….21 3.2.4. RNAse A treatment of DNA preparations…………………………………………….…..21
3.2.5. Synthesis of cDNA……………………………...……………………………………...……21
3.2.6. Primer extension analysis……………………………………………………………..……21
3.2.7. 5‘ and 3‘ RACE reactions…….…………………..……………………………….………..22
3.2.8. Polymerase chain reaction (PCR)………………………………………………….……...23
3.2.9. Sequencing of DNA………………………………………………………………….………23
3.2.10. Gelelctrophoresis of nucleic acids………………………………………………….……...23
3.2.11. Southern analysis of DNA…………………………………………………………….…….23
3.2.12. Northern analysis of RNA…………………………
3.2.13. RNA ligation………………………………………………………………….……24
3.2.14. Preparation of radiolabeled probes………………………….……….25
3.2.14.1. Radioactive labeling of PCR products…………………………………………………...25
3.2.14.2. Radioactive labeling of oligodesoxynucleotides…………………………………….….25
3.2.14.3. Preparation of radiolabeled RNA probes……………………………………..25
3.3. Hybridisation procedure…………………………………………………………………….26
3.4. Dot-blot DNA/RNA hybridisation analysis…………………………...26
II. Array Preparation…………………………………………………………………………...27
3.5. Nylon filter array preparation………………………………………………………………27
3.6. End-labeling of plastid transcripts…………………………27
3.7. Plastid run-on transcription assays……………………………………………………….27
3.8. Array hybridisation………………………………………….27
3.9. Image analysis……………………………………………………………………28
III. Manipulation of proteins……………………………………………………………………28
3.10. Extraction of leaf proteins………………………………………………………………….28
3.11. One-dimensional SDS-polyacrylamide gel electrophoresis…………………28
3.12. Silver staining of protein gels……………………………………………..……………….29
3.13. Coomasie Blue R-250 staining of protein gels………………………………..29
3.14. Electrophoretic transfer of proteins onto nitrocellulose membranes
(Western analysis)…………………………………………………………………….…….30
3.15. Immunological probing of proteins on nitrocellulose membranes……………..….……30
3.16. Immunological detection using enhanced chemiluminiscence………………………....30
IV. Bacterial transformation………………………………………………………….31
3.17. Preparation of competent bacteria…………………….………………….…….31
3.18. Transformation of bacteria cells…………………………………………………31
V. Manipulation of plant cells and organelles……………………………………..32
3.19. Seed sterilisation…………………………………………………………….……32
3.20. Isolation of plastids………………………………………….32
3.21. Fractionation of chloroplasts into stroma and thylakoid membranes………………….33
3.22. Isolation of the major thylakoid protein complexes……………………………………...34
3.23. Plastid transformation………………………………………………………………………34 3.24. Selection and regeneration of transplastomic mutants…………………………………35
3.25. Isolation of protoplasts and transformation for transient expression……….35

4. Results…………………………………………………………………………………….37

4.1. Identification of tobacco NEP genes……………………………………………….……...37
4.1.1. Characterisation and analysis of Nicotiana rpoT cDNAs…………….……….38
4.1.2. Subcellular localization of tobacco RpoT proteins……………………………………….41
4.2. Comparative analysis of plastid transcription profiles attributed to wild-type
and PEP-deficient transcription machineries……………..………………………………45
4.2.1. Plastid macroarrays and their limitations………………………………………………....48
4.2.2. Technical advances of array studies……………………………………………………...46
4.2.2.1. Specificity test………………………………………………………………………………..46
4.2.2.2. Sensitivity test………………………………….….47
4.2.2.3. Single-stranded vs. double-stranded probes……………………………………………..47
4.2.2.4. Probe size, blot and hybridisation reproducibility………………......49
4.2.2.5. What is the best probe for hybridising macroarrays?…………………………………....50
4.2.2.6. T4 Polynucleotide kinase specificity………………………………………………….……51
4.3. Comparison of expression profiles of wild-type and PEP-deficient plastids……….….52
4.3.1. Genes encoding photosynthesis-related components……………………………….….53
4.3.1.1. Transcriptional activity in wild-type and ∆rpoA mutant plastids………..….…53
4.3.1.2. Transcript levels in wild-type and ∆rpoA mutant plastids……………………….….…...58
4.3.1.3. Qualitative differences between wild-type and ∆rpoA plastid transcripts………….…..59
4.3.1.4. Protein accumulation in wild-type and ∆rpo mutant plastids……………………………62
4.3.2. Genes encoding components of the genetic apparatus………………………………...63
4.3.3. Heterogenic operons encoding components of both, the photosynthesis and
genetic apparatus…………………………………………………………………………....64
4.3.4. Genes specifying other functions……………………………………………………….….66
4.3.5. Open reading frames…………………………………….….67
4.3.6. Accumulation of aberrant transcripts of the rpoB-operon in ∆rpoA mutants…..……...72
4.4. PEP promoter studies………………………………………………………………………74
4.4.1. Cloning strategy and plastid transformation studies………………………………….…75
4.4.2. Analysis of rpoB promoter transformants……………………….…..76
4.4.2.1. Homoplastomy check of transformed lines………………….…………………………...76
4.4.2.2. Phenotype characterisation of rpoB promoter mutants…………………………………78
4.4.2.3. Transcriptional characterisation of the rpoB promoter mutants……………….……….79
4.5. Expression of genes for components of the translational machinery………………….82
4.5.1. n of tRNAs in tobacco plastids…………………………………………………82
4.5.2. Expression of genes coding for ribosomal subunits in tobacco wild-type and
PEP-deficient cells………………………………………………………………………….86 4.5.3. Expression of the rRNA operon…………………………..…………………………….….88
4.6. Analysis of the biogenesis of the cytochrome b /f complex……………..……90 6
4.6.1. Cloning strategy and homoplastomicity check of transformed pet-deficient
lines………………..………………………………………………………………………….91
4.6.2. Phenotype characterisation of transplastomic mutant lines……………….…97
4.7. Protein analysis of transplastomic mutant lines…………………………...……………..99
4.7.1. Complex formation…………………………………………………………….….99
4.7.2. Immunological characterisation of transplastomic lines………………….………….…100

5. Discussion……………………………………………………………………………....106

5.1. Conserved rpoT genes of both parental lines of tobacco………………………….…..106