Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2003
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2003 |
| Beschreibung: | VIII, 79 S. Ill., graph. Darst. |
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| 100 | 1 | |a Tsai, Chyn-Bey |d 1973- |e Verfasser |0 (DE-588)124771300 |4 aut | |
| 245 | 1 | 0 | |a Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris |c von Chyn-Bey Tsai |
| 264 | 1 | |c 2003 | |
| 300 | |a VIII, 79 S. |b Ill., graph. Darst. | ||
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| 650 | 0 | 7 | |a Nitratreduktase |g NADH |0 (DE-588)4348805-5 |2 gnd |9 rswk-swf |
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Datensatz im Suchindex
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| adam_text |
CONTENTS |
SUMMARY
-TT.T.TT._T.
,TV
ZUSAMMENFASSUNG_
VI I
1. I NTROD UCTION_ 1
1.1 OVERVIEW OF NITRATE ASSIMILATION IN HIGHER PLANTS.1
1.2 OVERVIEW OF NITRATE REDUCTASE.1
1.3 REGULATION OF NR.2
1.3.1 TRANSCRIPTIONAL REGULATION OF NR.3
1.3.1.1 BY NITRATE.3
1.3.1.2 BY LIGHT AND CARBOHYDRATES.4
1.3.2 POST-TRANSCRIPTIONAL REGULATION OF NR.5
1.3.2.1 BY NITRATE. 5
1.3.2.2 BY LIGHT AND CARBOHYDRATES.6
1.3.3 MECHANISM OF THE POST-TRANSLATIONAL REGULATION OF NR IN HIGHER
PLANTS
.6
1.3.4 THE ROLE OF THE N-TERMINAL DOMAIN IN NR REGULATION.8
1.4 AN EXCEPTIONAL EXAMPLE FROM RICINUS.9
1.5 OBJECTIVE OF THIS THESIS.10
2 RESULTS. 13
2.1 MOLECULAR CLONING OF NR GENE FROM RICINUS.13
2.1.1 THE PRIMARY RICINUS NR GENE SEQUENCE OBTAINED BY RT-PCR.13
2.1.2 THE FULL-LENGTH RICINUS NR GENE.14
2.1.3 ANALYSIS OF THE DEDUCED AMINO ACIDS SEQUENCE.15
2.2 HETEROLOGOUS EXPRESSION OF RCNR IN
PICHIA PASTOR IS.17
2.2.1 CONSTRUCTION.17
2.2.2 TRANSFORMATION.20
2.2.3 EXPRESSION.21
2.3 CHARACTERIZATION OF RECOMBINANT RICINUS NR.24
2.3.1 MODULATION OF NRS FROM PLANT LEAVES.24
2.3.2 MODULATION OF NRS FROM YEAST CELLS.25
2.3.3 RCNR HAS A HIGH MG
2+-SENSITIVITY WHEN MIXED WITH NR-FREE EXTRACTS
FROM RICINUS LEAVES AT PH 7.6, BUT NOT AT PH 6.5.27
2.3.4 PROTEIN FRACTIONATION BY AMMONIUM SULFATE.28
3 DISCUSSION. 31
3.1 DEDUCED PROTEIN SEQUENCE OF RICINUS NR.31
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/968463495
CONTENTS
II
3.1.1 THE N-TERMINUS.31
3.1.2 THE MOCO-BINDING REGION.32
3.1.3 THE HINGE 1 REGION.32
3.2 HETEROLOGOUS EXPRESSION OF RICINUS NR.33
3.3 CHARACTERIZATION OF RECOMBINANT RCNR.33
3.3.1
IN VITRO INACTIVATION BY ATP.33
3.3.2 SPECIFIC PROTEIN FACTOR(S) FOR HIGH MG
2+-SENSITIVITY OF RCNR.34
3.3.3 POSSIBLE REGULATORY FACTORS OF NR.35
3.4 THE COMPARISON OF RICINUS AND SQUASH NITRATE REDUCTASE.36
4 MATERIALS AND METHODS.39
4.1 PLANT MATERIAL.39
4.2 RNA PROTOCOLS.39
4.2.1 TOTAL RNA ISOLATION.39
4.2.2 ELECTROPHORESIS OF RNA.40
4.3 DNA PROTOCOLS.41
4.3.1 ISOLATION OF PLASMID-DNA FROM ESCHERICHIA COLI.41
4.3.2 RESTRICTION ENDONUCLEASES DIGESTION.41
4.3.3 ELECTROPHORESIS OF DNA.41
4.4 SEQUENCING.42
4.5 MOLECULAR CLONING OF RICINUS NR BY RT-PCR.42
4.6 AMPLIFICATION OF FULL-LENGTH RICINUS NR CDNA BY RACE.44
4.7
PICHIAPASTORIS EXPRESSION SYSTEM.45
4.8 PROTEIN PROTOCOLS.47
4.8.1 PROTEIN DETERMINATION.47
4.8.2 SDS-POLYACRYLAMIDE GELELECTROPHORESIS (SDS-PAGE).47
4.8.3 WESTERN BLOT AND IMMUNODETECTION OF NR.48
4.8.4 AMMONIUM SULFATE FRACTIONATION.48
4.9 NR ACTIVITY MEASUREMENTS.49
4.9.1 DETERMINATION OF NR ACTIVITY IN YEAST CELLS
(IN VIVO).49
4.9.2 ASSAY OF NR ACTIVITY
IN VITRO.49
4.9.3 ATP-DEPENDENT INACTIVATION
IN VITRO.50
4.10 GAS PHASE NO MEASUREMENTS.51
4.11 LIST OF CHEMICALS.53
CONTENTS
III
5 REFERENCES.
.55
^ APPENDIX 65
6.1 ABBREVIATIONS.65
6.2 OLIGO NUCLEOTIDE PRIMERS.66
6.3 VECTORS.67
6.3.1 PCR 2.1-TOPO.67
6.3.2 PPICZA.68
6.3.3 PHILD2.69
6.4 KEY INVARIANT RESIDUES IN RICINUS NR.70
6.5 AMINO ACID SEQUENCE ALIGNMENTS (N-TERMINUS).71
6.6 AMINO ACID SEQUENCE ALIGNMENTS (WITHOUT N-TERMINUS).72
6.7 PHYLOGENIES ANALYSIS (FULL-LENGTH NRS).78
6.8 PHYLOGENIES ANALYSIS (N-TERMINUS OF NRS).79 |
| any_adam_object | 1 |
| author | Tsai, Chyn-Bey 1973- |
| author_GND | (DE-588)124771300 |
| author_facet | Tsai, Chyn-Bey 1973- |
| author_role | aut |
| author_sort | Tsai, Chyn-Bey 1973- |
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| building | Verbundindex |
| bvnumber | BV017183778 |
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| genre_facet | Hochschulschrift |
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| illustrated | Illustrated |
| indexdate | 2024-11-22T17:37:19Z |
| institution | BVB |
| language | English |
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| oclc_num | 643085804 |
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| owner | DE-20 DE-12 |
| owner_facet | DE-20 DE-12 |
| physical | VIII, 79 S. Ill., graph. Darst. |
| publishDate | 2003 |
| publishDateSearch | 2003 |
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| spelling | Tsai, Chyn-Bey 1973- Verfasser (DE-588)124771300 aut Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris von Chyn-Bey Tsai 2003 VIII, 79 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2003 Pichia pastoris (DE-588)4407211-9 gnd rswk-swf Rizinus (DE-588)4254423-3 gnd rswk-swf Nitratreduktase (DE-588)4324240-6 gnd rswk-swf Nitratreduktase NADH (DE-588)4348805-5 gnd rswk-swf Heterologe Genexpression (DE-588)4342015-1 gnd rswk-swf Enzymatische Regulation (DE-588)4136839-3 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Rizinus (DE-588)4254423-3 s Nitratreduktase (DE-588)4324240-6 s Enzymatische Regulation (DE-588)4136839-3 s DE-604 Nitratreduktase NADH (DE-588)4348805-5 s Heterologe Genexpression (DE-588)4342015-1 s Pichia pastoris (DE-588)4407211-9 s DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010355172&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Tsai, Chyn-Bey 1973- Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris Pichia pastoris (DE-588)4407211-9 gnd Rizinus (DE-588)4254423-3 gnd Nitratreduktase (DE-588)4324240-6 gnd Nitratreduktase NADH (DE-588)4348805-5 gnd Heterologe Genexpression (DE-588)4342015-1 gnd Enzymatische Regulation (DE-588)4136839-3 gnd |
| subject_GND | (DE-588)4407211-9 (DE-588)4254423-3 (DE-588)4324240-6 (DE-588)4348805-5 (DE-588)4342015-1 (DE-588)4136839-3 (DE-588)4113937-9 |
| title | Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris |
| title_auth | Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris |
| title_exact_search | Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris |
| title_full | Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris von Chyn-Bey Tsai |
| title_fullStr | Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris von Chyn-Bey Tsai |
| title_full_unstemmed | Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris von Chyn-Bey Tsai |
| title_short | Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris |
| title_sort | molecular cloning and characterization of nitrate reductase from ricinus communis l heterologously expressed in pichia pastoris |
| topic | Pichia pastoris (DE-588)4407211-9 gnd Rizinus (DE-588)4254423-3 gnd Nitratreduktase (DE-588)4324240-6 gnd Nitratreduktase NADH (DE-588)4348805-5 gnd Heterologe Genexpression (DE-588)4342015-1 gnd Enzymatische Regulation (DE-588)4136839-3 gnd |
| topic_facet | Pichia pastoris Rizinus Nitratreduktase Nitratreduktase NADH Heterologe Genexpression Enzymatische Regulation Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010355172&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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