Functional anatomy of neurospora crassa kinesin: mutational analysis, expression of subdomains and the search for a binding partner
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
1999
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 116 S. Ill., graph. Darst. |
Internformat
MARC
| LEADER | 00000nam a2200000 c 4500 | ||
|---|---|---|---|
| 001 | BV012804469 | ||
| 003 | DE-604 | ||
| 005 | 20030228 | ||
| 007 | t | ||
| 008 | 991011s1999 ad|| m||| 00||| eng d | ||
| 016 | 7 | |a 957753756 |2 DE-101 | |
| 035 | |a (OCoLC)247361011 | ||
| 035 | |a (DE-599)BVBBV012804469 | ||
| 040 | |a DE-604 |b ger |e rakwb | ||
| 041 | 0 | |a eng | |
| 049 | |a DE-19 |a DE-29T |a DE-12 |a DE-355 |a DE-188 | ||
| 100 | 1 | |a Kirchner, Joachim |d 1967- |e Verfasser |0 (DE-588)121520498 |4 aut | |
| 245 | 1 | 0 | |a Functional anatomy of neurospora crassa kinesin |b mutational analysis, expression of subdomains and the search for a binding partner |c von Joachim Kirchner |
| 264 | 1 | |c 1999 | |
| 300 | |a 116 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a München, Univ., Diss., 1999 | ||
| 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=008708703&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-008708703 | |
Datensatz im Suchindex
| _version_ | 1807504926802182144 |
|---|---|
| adam_text |
CONTENTS
ABBREVIATIONS
6
1
INTRODUCTION
9
1.1
MOLECULAR
MOTORS
AND
THE
CYTOSKELETON
.
9
1.2
CONVENTIONAL
KINESINS
.
11
1.3
THE
NEUROSPORA
CRASSA
KINESIN
NULL
MUTANT
.
12
1.4
GOALS
OF
THE
PRESENT
WORK
.
15
2
MATERIALS
AND
METHODS
16
2.1
MATERIALS
.
16
2.2
VECTORS
AND
STRAINS
.
16
2.2.1
VECTORS
.
16
2.2.2
BACTERIAL
STRAINS
.
17
2.2.3
CULTIVATION
OF
E.
COLL
.
17
2.2.4
N.
CRASSA
STRAINS
.
17
2.2.5
CULTIVATION
OF
NEUROSPORA
CRASSA
.
17
2.3
DNA
METHODS
.
18
2.3.1
AGAROSE
GEL
ELECTROPHORESIS
.
18
2.3.2
DNA
EXTRACTION
FROM
AGAROSE
GELS
.
19
2.3.3
DETERMINATION
OF
DNA
CONCENTRATION
.
19
2.3.4
PREPARATION
OF
PLASMID
DNA
.
19
2.3.5
PHENOL
EXTRACTION
AND
PRECIPITATION
OF
DNA
.
19
2.3.6
DNA
CLEAVAGE
WITH
RESTRICTION
ENZYMES
.
19
2.3.7
PARTIAL
RESTRICTION
DIGEST
.
20
2.3.8
LIGATION
OF
DNA
INTO
A
PLASMID
VECTOR
.
20
2.3.9
PREPARATION
OF
ELECTROCOMPETENT
CELLS
.
20
2.3.10
ELECTROTRANSFORMATION
OF
E.
COLI
.
20
2.3.11
IDENTIFICATION
OF
TRANSFORMED
CLONES
IN
E.
COLI
.
21
2.3.12
SEQUENCING
OF
DNA
.
21
CONTENTS
2
2.3.13
POLYMERASE
CHAIN
REACTION
.
21
2.3.14
INTRODUCTION
OF
POINT
MUTATIONS
.
22
2.3.15
OLIGONUCLEOTIDES
.
22
2.3.15.1
VECTOR
PRIMERS
.
22
2.3.15.2
SEQUENCING
PRIMERS
.
23
2.3.15.3
PRIMERS
FOR
C-TERMINAL
DELETION
CONSTRUCTS
.
.
23
2.3.15.4
PRIMERS
FOR
INTERNAL
DELETION
CONSTRUCTS
.
.
.
24
2.3.15.5
PRIMERS
FOR
CHIMAERAS
.
25
2.3.15.6
PRIMERS
FOR
POINT
MUTATIONS
.
25
2.3.15.7
PRIMERS
FOR
C-TERMINAL
FRAGMENTS
.
26
2.3.16
GENERATION
OF
CONSTRUCTS
FOR
TRANSFORMATION
.
26
2.3.16.1
CONSTRUCT
NOMENCLATURE
.
26
2.3.16.2
NCKIN
C-TERMINAL
DELETION
MUTANTS
.
.
27
2.3.16.3
INTERNAL
DELETION
MUTANTS
.
27
2.3.16.4
POINT
MUTATIONS
.
27
2.3.16.5
CHIMAERAS
.
27
2.3.16.6
BACTERIAL
C-TERMINAL
DELETION
CONSTRUCTS
.
29
2.3.16.7
C-TERMINAL
CONSTRUCTS
.
29
2.4
BIOCHEMICAL
METHODS
.
30
2.4.1
SDS-POLYACRYLAMIDE
GELELECTROPHORESIS
(PAGE)
.
30
2.4.2
COOMASSIE
STAINING
.
.'
.
30
2.4.3
COLLOIDAL
COOMASSIE
STAINING
.
30
2.4.4
SILVER
STAINING
OF
PROTEINS
.
31
2.4.5
DETERMINATION
OF
PROTEIN
CONCENTRATION
.
31
2.4.6
PROTEIN
PURIFICATION
.
31
2.4.6.1
PURIFICATION
OF
TUBULIN
.
31
2.4.6.2
PURIFICATION
OF
NCKIN
OR
KINESIN
CONSTRUCTS
FROM
N.
CRASSA
.
33
2.4.6.3
PURIFICATION
OF
RECOMBINANT
NCKIN
CONSTRUCTS
FROM
E.
COH
.
34
2.4.6.4
ISOLATION
OF
RECOMBINANT
NCKIN
CONSTRUCTS
WITH
A
PHOSPHOCELLULOSE-COLUMN
.
34
2.4.6.5
PURIFICATION
OF
HISTIDINE-TAGGED
CONSTRUCTS
.
.
35
2.4.6.6
PURIFICATION
OF
GST-FUSION
CONSTRUCTS
.
36
2.4.7
POLYMERIZATION
OF
MICROTUBULES
M
VITRO
.
36
2.4.8
AFFINITY
CHROMATOGRAPHY
.
37
2.4.8.1
AFFINITY
CHROMATOGRAPHY
WITH
MT-BOUND
KI
NESIN
CONSTRUCTS
.
37
CONTENTS
3
2.4.8.2
AFFINITY
CHROMATOGRAPHY
WITH
MATRIX-BOUND
HIS-TAG
OR
GST-FUSION
CONSTRUCTS
.
37
2.4.9
GEL
FILTRATION
BINDING
ASSAY
.
37
2.4.10
DENSITY
GRADIENT
CENTRIFUGATION
AND
GEL
FILTRATION
.
37
2.4.11
ANTIBODIES
.
39
2.4.11.1
CROSSLINKING
OF
PEPTIDES
TO
KLH
.
39
2.4.11.2
IMMUNIZATION
AND
PREPARATION
OF
ANTISERUM
.
.
39
2.4.11.3
DETECTION
OF
PROTEINS
BY
WESTERN
BLOT
.
39
2.4.11.4
AFFINITY
PURIFICATION
WITH
NITROCELLULOSE-STRIPS
.
40
2.4.12
ATPASE
ASSAY
.
40
2.4.12.1
COUPLED
ATPASE
ASSAY
.
40
2.4.12.2
DETERMINATION
OF
MOTOR
CONCENTRATION
.
42
2.4.12.3
CALCULATIONS
FOR
THE
ATPASE
ASSAY
.
42
2.5
SEQUENCE
ANALYSIS
.
43
2.6
N.
CRASSA
METHODS
.
43
2.6.1
PREPARATION
OF
PEG-COMPETENT
SPHAEROPLASTS
.
43
2.6.2
TRANSFORMATION
OF
N.
CRASSA
.
44
2.6.3
CHARACTERIZATION
OF
FUNGAL
CLONES
CONTAINING
MUTANT
KINESINS
.
44
2.6.4
DETERMINATION
OF
KINESIN
EXPRESSION
LEVELS
IN
POSITIVE
CLONES
.
45
2.7
ORGANELLE
METHODS
.
46
2.7.1
ISOLATION
OF
MICROSOMAL
FRACTIONS
FROM
N.
CRASSA
.
46
2.7.2
ISOLATION
OF
MITOCHONDRIAL
FRACTIONS
FROM
N.
CRASSA
.
46
2.7.3
FRACTIONATION
OF
ORGANELLES
IN
A
SUCROSE
GRADIENT
.
46
2.7.4
COISOLATION
OF
MOTOR
MOLECULES
AND
ORGANELLES
.
46
2.7.5
IN
VITRO
ATTACHMENT
OF
MOTORS
TO
ORGANELLES
.
47
2.7.6
SOLUBILIZATION
OF
ORGANELLES
WITH
DETERGENT
.
47
2.8
MICROSCOPY
METHODS
.
47
2.8.1
VIDEO-ENHANCED
LIGHT
MICROSCOPY
.
47
2.8.2
MOTILITY
ASSAY
WITH
MOTOR
ATTACHED
TO
THE
COVERSLIP
.
.
47
2.8.3
POLARITY
DETERMINATION
OF
ISOLATED
MOTOR
.
48
2.8.4
BEAD
MOTILITY
ASSAY
.
48
2.8.5
ORGANELLE
MOTILITY
ASSAY
.
48
3
RESULTS
49
3.1
SEQUENCE
OF
THE
NCKIN
GENE
.
49
3.2
FUNGAL
VS.
ANIMAL
KINESINS:
A
SEQUENCE
COMPARISON
.
56
3.3
MUTANT
KINESIN
CONSTRUCTS
IN
E.
COLT
.
62
CONTENTS
4
3.3.1
EXPRESSION
OF
MUTANT
KINESIN
CONSTRUCTS
IN
E.
COLT
.
.
62
3.3.2
PROPERTIES
OF
RECOMBINANT
KINESIN
CONSTRUCTS
.
64
3.4
GENERATION
OF
AN
ANTISERUM
AGAINST
A
C-TERMINAL
PEPTIDE
FROM
NCKIN
.
66
3.5
IN
VIVO
MUTATIONAL
ANALYSIS
OF
NCKIN
.
66
3.5.1
SUPPRESSION
OF
THE
GROWTH
DEFECT
OF
THE
NEUROSPORA
CRASSA
KINESIN
NULL
MUTANT
.
66
3.5.2
C-TERMINAL
DELETION
MUTANTS
OF
NCKIN
.
68
3.5.3
EXPRESSION
OF
NHKIN
IN
THE
NCKIN
NULL
MUTANT
.
71
3.5.4
C-TERMINAL
CHIMAERAS
BETWEEN
NCKIN
AND
OTHER
FUNGAL
KINESINS
.
71
3.5.5
CONSTRUCTS
MUTATED
IN
THE
STALK
REGION
.
74
3.6
EXPRESSION
LEVELS
OF
MUTANT
KINESIN
CONSTRUCTS
.
75
3.7
EXPRESSION
OF
POINT
MUTATED
CONSTRUCTS
IN
THE
NCKIN
NULL
MUTANT
78
3.8
EXPRESSION
OF
C-TERMINAL
FRAGMENTS
OF
NCKIN
.
78
3.8.1
EXPRESSION
OF
HISTIDINE-TAGGED
CONSTRUCTS
.
78
3.8.2
EXPRESSION
OF
C-TERMINAL
CONSTRUCTS
IN
N.
CRASSA
.
79
3.8.3
EXPRESSION
OF
GST
FUSION
CONSTRUCTS
.
81
3.9
INTERACTION
OF
THE
C-TERMINUS
OF
NCKIN
WITH
NC
YY
740
.
81
3.10
THE
SEARCH
FOR
A
NCKIN
CARGO
RECEPTOR
.
82
3.10.1
ATTACHMENT
OF
NCKIN
TO
NEUROSPORA
ORGANELLES
.'.
82
3.10.2
ORGANELLE
MOTILITY
ASSAY
.
85
3.10.3
INCUBATION
OF
N.
CRASSA
EXTRACTS
WITH
MICROTUBULE-BOUND
MOTOR
.
85
3.10.4
INCUBATION
OF
N.
CRASSA
EXTRACTS
WITH
HIS-NKCF
858
BOUND
TO
A
TALON
RESIN
.
85
3.10.5
INCUBATION
OF
N.
CRASSA
EXTRACTS
WITH
GST
FUSION
CON
STRUCTS
.
86
4
DISCUSSION
88
4.1
EXPRESSION
AND
PROPERTIES
OF
RECOMBINANT
NCKIN
CONSTRUCTS
.
.
88
4.2
MUTATIONAL
ANALYSIS
OF
THE
NCKIN
TAIL
AND
STALK
.
89
4.2.1
INFLUENCE
OF
EXPRESSION
LEVELS
ON
SUPPRESSION
OF
THE
NULL
PHENOTYPE
.
94
4.3
FUNGAL
VS.
ANIMAL
KINESINS
.
94
4.4
THE
SEARCH
FOR
A
KINESIN
RECEPTOR
.
98
4.5
FUTURE
PROSPECTS
.
99
CONTENTS
5
5
SUMMARY
100
5.1
SUMMARY
.
100
5.2
ZUSAMMENFASSUNG
.
102
6
REFERENCES
104
ACKNOWLEDGEMENTS
113
CURRICULUM
VITAE
115 |
| any_adam_object | 1 |
| author | Kirchner, Joachim 1967- |
| author_GND | (DE-588)121520498 |
| author_facet | Kirchner, Joachim 1967- |
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| building | Verbundindex |
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| ctrlnum | (OCoLC)247361011 (DE-599)BVBBV012804469 |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV012804469 |
| illustrated | Illustrated |
| indexdate | 2024-08-16T01:17:05Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-008708703 |
| oclc_num | 247361011 |
| open_access_boolean | |
| owner | DE-19 DE-BY-UBM DE-29T DE-12 DE-355 DE-BY-UBR DE-188 |
| owner_facet | DE-19 DE-BY-UBM DE-29T DE-12 DE-355 DE-BY-UBR DE-188 |
| physical | 116 S. Ill., graph. Darst. |
| publishDate | 1999 |
| publishDateSearch | 1999 |
| publishDateSort | 1999 |
| record_format | marc |
| spelling | Kirchner, Joachim 1967- Verfasser (DE-588)121520498 aut Functional anatomy of neurospora crassa kinesin mutational analysis, expression of subdomains and the search for a binding partner von Joachim Kirchner 1999 116 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 1999 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=008708703&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Kirchner, Joachim 1967- Functional anatomy of neurospora crassa kinesin mutational analysis, expression of subdomains and the search for a binding partner 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 | Functional anatomy of neurospora crassa kinesin mutational analysis, expression of subdomains and the search for a binding partner |
| title_auth | Functional anatomy of neurospora crassa kinesin mutational analysis, expression of subdomains and the search for a binding partner |
| title_exact_search | Functional anatomy of neurospora crassa kinesin mutational analysis, expression of subdomains and the search for a binding partner |
| title_full | Functional anatomy of neurospora crassa kinesin mutational analysis, expression of subdomains and the search for a binding partner von Joachim Kirchner |
| title_fullStr | Functional anatomy of neurospora crassa kinesin mutational analysis, expression of subdomains and the search for a binding partner von Joachim Kirchner |
| title_full_unstemmed | Functional anatomy of neurospora crassa kinesin mutational analysis, expression of subdomains and the search for a binding partner von Joachim Kirchner |
| title_short | Functional anatomy of neurospora crassa kinesin |
| title_sort | functional anatomy of neurospora crassa kinesin mutational analysis expression of subdomains and the search for a binding partner |
| title_sub | mutational analysis, expression of subdomains and the search for a binding partner |
| 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=008708703&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT kirchnerjoachim functionalanatomyofneurosporacrassakinesinmutationalanalysisexpressionofsubdomainsandthesearchforabindingpartner |