Crosslinking studies on the conventional kinesin of Neurospora crassa:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2004
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | München, Univ., Diss., 2004 |
| Beschreibung: | VI, 109 Bl. Ill., graph. Darst. |
Internformat
MARC
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| 007 | t | ||
| 008 | 050113s2004 gw ad|| m||| 00||| eng d | ||
| 015 | |a 0501,H,0974 |2 dnb | ||
| 016 | 7 | |a 972646299 |2 DE-101 | |
| 035 | |a (OCoLC)76550808 | ||
| 035 | |a (DE-599)BVBBV019656998 | ||
| 040 | |a DE-604 |b ger |e rakddb | ||
| 041 | 0 | |a eng | |
| 044 | |a gw |c XA-DE | ||
| 049 | |a DE-12 |a DE-19 | ||
| 084 | |a 570 |2 sdnb | ||
| 100 | 1 | |a Hahlen, Katrin |d 1972- |e Verfasser |0 (DE-588)129476757 |4 aut | |
| 245 | 1 | 0 | |a Crosslinking studies on the conventional kinesin of Neurospora crassa |c vorgelegt von Katrin Hahlen |
| 264 | 1 | |c 2004 | |
| 300 | |a VI, 109 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a München, Univ., Diss., 2004 | ||
| 650 | 0 | 7 | |a Neurospora crassa |0 (DE-588)4171597-4 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Kinesin |0 (DE-588)4236029-8 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Neurospora crassa |0 (DE-588)4171597-4 |D s |
| 689 | 0 | 1 | |a Kinesin |0 (DE-588)4236029-8 |D s |
| 689 | 0 | |5 DE-604 | |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012985498&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-012985498 | |
Datensatz im Suchindex
| _version_ | 1807864726914334720 |
|---|---|
| adam_text |
CONTENTS
I
CONTENTS
ABBREVIATIONS.
V
1
INTRODUCTION
.
1
1.1
MOLECULAR
MOTORS
.
1
1.1.1
KINESIN
SUPERFAMILY
.
2
1.1.2
SIMILARITIES
BETWEEN
KINESINS,
MYOSINS
AND
G-PROTEINS
.
3
1.2
CONVENTIONAL
KINESINS
.
4
1.2.1
FUNCTIONAL
ANATOMY
.
4
1.2.2
MODEL
OF
MOTION
.
6
1.3
NEUROSPORA
CRASSA
KINESINS
.
7
1.4
GOALS
OF
THE
PRESENT
WORK
.
8
2
MATERIALS
AND
METHODS.
11
2.1
MATERIALS
.
11
2.1.1
REAGENTS
AND
OTHER
MATERIALS
.
11
2.1.2
VECTORS
.
11
2.1.3
BACTERIAL
STRAINS
.
11
2.1.4
MEDIA
AND
CULTIVATION
OF
E.
COLI
.
11
2.2
MOLECULAR
BIOLOGY
METHODS
.
12
2.2.1
AGAROSE
GEL
ELECTROPHORESIS
.
12
2.2.2
DNA
EXTRACTION
FROM
AGAROSE
GELS
.
12
2.2.3
DETERMINATION
OF
DNA
CONCENTRATION
.
12
2.2.4
PREPARATION
OF
PLASMID
DNA
.
12
2.2.5
DNA
CLEAVAGE
WITH
RESTRICTION
ENDONUCLEASES
.
12
2.2.6
LIGATION
OF
DNA
INTO
PLASMID
VECTORS
.
13
2.2.7
PREPARATION
AND
TRANSFORMATION
OF
COMPETENT
CELLS
.
13
2.2.7.1
PREPARATION
OF
ELECTROCOMPETENT
CELLS
.
13
2.2.7.2
ELECTROPORATION
.
13
2.2.7.3
PREPARATION
OF
SEM
COMPETENT
CELLS
.
13
2.2.7.4
HEAT
SHOCK
TRANSFORMATION
.
14
2.2.8
IDENTIFICATION
OF
TRANSFORMED
CLONES
IN
E.
COLI
.
14
2.2.9
POLYMERASE
CHAIN
REACTION
(PCR)
.
14
2.2.10
INTRODUCTION
OF
POINT
MUTATIONS
.
15
2.2.11
OLIGONUCLEOTIDES
.
15
2.2.11.1
PRIMERS
FOR
THE
REMOVAL
OR
INSERTION
OF
RESTRICTION
SITES
.
15
2.2.11.2
PRIMERS
FOR
THE
REMOVAL
OF
CYSTEINE
RESIDUES
38,
59
AND
307
.
15
CONTENTS
II
2.2.11.3
PRIMERS
FOR
THE
INSERTION
OF
CYSTEINE
RESIDUES
.
16
2.2.11.4
PRIMERS
FOR
THE
CONSTRUCTION
OF
MONOMERIC
CONSTRUCTS
.
17
2.2.11.5
PRIMER
FOR
THE
MONOMERIC
HUMAN
KINESIN
CONSTRUCT
.
17
2.2.11.6 SEQUENCING
PRIMERS
.
17
2.2.12
GENERATION
OF
CONSTRUCTS
.
17
2.2.13
SUMMARY
OF
ALL
MEASURED
CONSTRUCTS
.
21
2.3
BIOCHEMICAL
METHODS
.
22
2.3.1
SDS-POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(PAGE)
.
22
2.3.2
COOMASSIE
STAINING
.
23
2.3.3
COLLOIDAL
COOMASSIE
STAINING
.
23
2.3.4
EXPRESSION
OF
KINESIN
CONSTRUCTS
.
23
2.3.5
PROTEIN
PURIFICATION
.
23
2.3.5.1
PURIFICATION
OF
BACTERIALLY
EXPRESSED
NCKIN
.
23
2.3.5.2
PURIFICATION
OF
BACTERIALLY
EXPRESSED
HSKIN
.
24
2.3.5.3
PURIFICATION
OF
TUBULIN
.
25
2.3.6
DETERMINATION
OF
PROTEIN
CONCENTRATION
.
27
2.3.7
POLYMERISATION
OF
MICROTUBULES
.
27
2.3.8
DETERMINATION
OF
MICROTUBULE
CONCENTRATION
.
27
2.3.9
MICROSCOPY
.
28
2.3.9.1
VIDEO
ENHANCED
LIGHT
MICROSCOPY
.
28
2.3.9.2
MOTILITY
ASSAY
.
28
2.3.10
ATPASE
ASSAYS
.
29
2.3.10.1
BASAL
ATPASE
ASSAY
.
29
2.3.10.2
MICROTUBULE-ACTIVATED
ATPASE
ASSAY
.
30
2.3.10.3
CALCULATIONS
FOR
THE
ATPASE
ASSAY
.
31
2.3.11
MANTADP
EXPERIMENTS
.
32
2.3.11.1
LOADING
OF
KINESIN
MOTOR
DOMAINS
WITH
MANTADP
.
32
2.3.11.2 STOICHIOMETRY
OF
KINESIN-MANTADP
COMPLEXES
.
33
2.3.11.3
FLUOROMETRIC
MEASUREMENTS
OF
MANTADP
RELEASE
.
33
2.3.12 SEDIMENTATION
ASSAY
.
34
2.3.13
MEASUREMENTS
UNDER
REDUCING
AND
OXIDISING
CONDITIONS
.
34
3
RESULTS
.
36
3.1
RATIONALE
FOR
THE
DESIGN
OF
CROSSLINKING
MUTANTS
.
36
3.1.1
WILD-TYPE
CONSTRUCTS
.
37
3.1.2
NECK/NECK
CROSSLINKING
CONSTRUCTS
.
38
3.1.3
NECK-LINKER/MOTOR
CORE
CROSSLINKING
CONSTRUCTS
.
39
3.1.3.1
NCKIN
.
39
3.1.3.2
HSKIN
.
40
CONTENTS
III
3.1.4
A4/A6
CROSSLINKING
CONSTRUCTS
.
40
3.2
PURIFICATION
OF
BACTERIALLY
EXPRESSED
KINESIN
.
41
3.3
PROOF
OF
THE
CROSSLINK
.
43
3.3.1
WILD-TYPE
CONSTRUCTS
.
45
3.3.2
NECK/NECK
CROSSLINKING
CONSTRUCTS
.
45
3.3.3
NECK-LINKER/MOTOR
CORE
CROSSLINKING
CONSTRUCTS
.
46
3.3.4
A4/A6
CROSSLINKING
CONSTRUCTS
.
47
3.4
MOTILITY
AND
ATPASE
MEASUREMENTS
.
47
3.4.1
WILD-TYPE
CONSTRUCTS
.
49
3.4.1.1
MOTILITY
.
49
3.4.1.2
ATPASE
ACTIVITY
.
49
3.4.1.3
SUMMARY
OF
NCKINWT
.
50
3.4.2
NECK/NECK
CROSSLINKING
CONSTRUCTS
.
50
3.4.2.1
MOTILITY
.
50
3.4.2.2
ATPASE
ACTIVITY
.
51
3.4.2.3
SUMMARY
OF
NCKIN_S341C
AND
NCKIN_P342C
.
52
3.4.3
NECK-LINKER/MOTOR
CORE
CROSSLINKING
CONSTRUCTS
.
52
3.4.3.1
NCKIN_S3C_A334C
.
52
3.4.3.1.1
MOTILITY
.52
3.4.3.1.2
ATPASE
ACTIVITY
.
53
3.4.3.1.3
SUMMARY
OF
NCKIN_S3C_A334C
.
55
3.4.3.2
NCKIN_A226C_E339C
.
56
3.4.3.2.1
MOTILITY
.
56
3.4.3.2.2
ATPASE
ACTIVITY
.56
3.4.3.2.3
SUMMARY
OF
NCKIN_A226C_E339C
.
58
3.4.3.3
HSKIN_K222C_E334C
.
59
3.4.3.3.
1
ATPASE
ACTIVITY
.59
3.4.3.3.2
SUMMARY
OF
HSKIN_K222C_E334C
.
60
3.4.4
A4/A6
CROSSLINKING
CONSTRUCTS
.
60
3.4.4.1
MOTILITY
.
60
3.4.4.2
ATPASE
ACTIVITY
.
61
3.4.4.3
SUMMARY
OF
NCKIN_T273C_S329C
.
63
3.4.5
SUMMARY
OF
ALL
CONSTRUCTS
.
64
3.5
COMPETITIVE
MOTILITY
ASSAYS
.
67
3.5.1
COMPETITION
OF
NCKIN433_WT
AND
NCKIN433_S3C_A334C
.
67
3.5.2
COMPETITION
OF
NCKIN433_WT
AND
NCKIN433_A226C_E339C
.
68
3.5.3
SUMMARY
OF
COMPETITIVE
MOTILITY
ASSAYS
.
68
3.6
SEDIMENTATION
ASSAYS
.
69
3.6.1
MICROTUBULE
AFFINITIES
OF
NCKIN433_A226C_E339C
.
71
CONTENTS
IV
3.6.2
MICROTUBULE
AFFINITIES
OF
NCKIN343_A226C_E339C
.
72
3.7
BASAL
ATPASE
MEASUREMENTS
.
73
3.7.1
NCKIN_WT
.
74
3.7.2
NCKIN433_S3C_A334C
.
74
3.7.3
NCKIN_A226C_E339C
.
74
3.8
ADP
RELEASE
EXPERIMENTS
.
76
3.8.1
NCKIN433_A226C_E339C
.
77
4
DISCUSSION
.
81
4.1
EVIDENCE
FOR
CROSSLINKING
.
81
4.2
VALIDITY
OF
WILD-TYPE
CONSTRUCTS
.82
4.3
NECK/NECK
CROSSLINKING
CONSTRUCTS
.82
4.3.1
CROSSLINKING
EFFICIENCY
.
82
4.3.2
CHARACTERISATION
OF
MOTILE
AND
HYDROLYSIS
PROPERTIES
.
83
4.3.3
COMPARISON
WITH
OTHER
NECK/NECK
CROSSLINKING
CONSTRUCTS
.
83
4.3.4
NECK
COILED-COIL
UNWINDING
HYPOTHESIS
.
84
4.4
NECK-LINKER/MOTOR
CORE
CROSSLINKING
CONSTRUCTS
.
85
4.4.1
CROSSLINKING
EFFICIENCY
.
85
4.4.2
CHARACTERISATION
OF
MOTILITY,
HYDROLYSIS
AND
BINDING
PROPERTIES
.
86
4.4.2.1
NCKIN_S3C_A334C
CONSTRUCTS
.
86
4.4.2.2 NCKIN_A226C_E339C
CONSTRUCTS
.
86
4.4.2.3
HSKIN_K222C_E334C
CONSTRUCTS
.
88
4.4.3
COMPARISON
OF
NECK-LINKER/MOTOR
CORE
CROSSLINKING
CONSTRUCTS
.
89
4.4.4
NECK-LINKER
UNZIPPERING
HYPOTHESIS
.
90
4.5
A4/A6
CROSSLINKING
CONSTRUCTS
.
93
4.5.1
CROSSLINKING
EFFICIENCY
.
93
4.5.2
CHARACTERISATION
OF
MOTILE
AND
HYDROLYSIS
PROPERTIES
.
93
4.5.3
ROTATION
OF
HELIX
A4
AGAINST
THE
MOTOR
CORE
.
94
4.6
FUTURE
PROSPECTS
.
95
5
SUMMARY
.
97
5.1
SUMMARY
.97
5.2
ZUSAMMENFASSUNG
.98
6
REFERENCES
.
100
ACKNOWLEDGEMENTS
.
108
CURRICULUM
VITAE
.
109 |
| any_adam_object | 1 |
| author | Hahlen, Katrin 1972- |
| author_GND | (DE-588)129476757 |
| author_facet | Hahlen, Katrin 1972- |
| author_role | aut |
| author_sort | Hahlen, Katrin 1972- |
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| building | Verbundindex |
| bvnumber | BV019656998 |
| ctrlnum | (OCoLC)76550808 (DE-599)BVBBV019656998 |
| discipline | Biologie |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV019656998 |
| illustrated | Illustrated |
| indexdate | 2024-08-20T00:35:55Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-012985498 |
| oclc_num | 76550808 |
| open_access_boolean | |
| owner | DE-12 DE-19 DE-BY-UBM |
| owner_facet | DE-12 DE-19 DE-BY-UBM |
| physical | VI, 109 Bl. Ill., graph. Darst. |
| publishDate | 2004 |
| publishDateSearch | 2004 |
| publishDateSort | 2004 |
| record_format | marc |
| spelling | Hahlen, Katrin 1972- Verfasser (DE-588)129476757 aut Crosslinking studies on the conventional kinesin of Neurospora crassa vorgelegt von Katrin Hahlen 2004 VI, 109 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 2004 Neurospora crassa (DE-588)4171597-4 gnd rswk-swf Kinesin (DE-588)4236029-8 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Neurospora crassa (DE-588)4171597-4 s Kinesin (DE-588)4236029-8 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012985498&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Hahlen, Katrin 1972- Crosslinking studies on the conventional kinesin of Neurospora crassa Neurospora crassa (DE-588)4171597-4 gnd Kinesin (DE-588)4236029-8 gnd |
| subject_GND | (DE-588)4171597-4 (DE-588)4236029-8 (DE-588)4113937-9 |
| title | Crosslinking studies on the conventional kinesin of Neurospora crassa |
| title_auth | Crosslinking studies on the conventional kinesin of Neurospora crassa |
| title_exact_search | Crosslinking studies on the conventional kinesin of Neurospora crassa |
| title_full | Crosslinking studies on the conventional kinesin of Neurospora crassa vorgelegt von Katrin Hahlen |
| title_fullStr | Crosslinking studies on the conventional kinesin of Neurospora crassa vorgelegt von Katrin Hahlen |
| title_full_unstemmed | Crosslinking studies on the conventional kinesin of Neurospora crassa vorgelegt von Katrin Hahlen |
| title_short | Crosslinking studies on the conventional kinesin of Neurospora crassa |
| title_sort | crosslinking studies on the conventional kinesin of neurospora crassa |
| topic | Neurospora crassa (DE-588)4171597-4 gnd Kinesin (DE-588)4236029-8 gnd |
| topic_facet | Neurospora crassa Kinesin Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012985498&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT hahlenkatrin crosslinkingstudiesontheconventionalkinesinofneurosporacrassa |