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UBC Theses and Dissertations
Isolation and functional studies of D. discoideum cyclin B gene during growth and development Luo, Qian Kathy
Abstract
Cyclin B plays an essential role i n cell cycle regulation by complexing with Cdc2 protein kinase to direct cells into mitosis. A cyclin B gene (cycB) has been isolated from D. discoideum. The cyclin box region of the protein encoded by cycB has a high degree of sequence identity with B-type cyclins of other species. During celldivision synchronized growth, levels of cyclin B mRNA and protein were found to oscillate during the cell cycle with maximum accumulation occurring prior to and during cell division. Northern blot analysis showed that the cycB mRNA levels peaked twice during development, once during early aggregation (3 h) and again at the tipped aggregate stage (12 h). In contrast, the levels of cyclin B protein and histone H I kinase activity of the Cdc2-immunoprecipitate increased only during early development and then gradually decreased as development continued. The N-terminal region of the cyclin B protein has the conserved protein degradation motif, typical of cyclin B proteins. The 5' portion of the D. discoideum cycB gene was deleted and the truncated gene was cloned downstream from the regulatable discoidin promoter. Overexpression of this truncated cyclin B gene elevated the histone H I kinase activity of the Cdc2-immunoprecipitates, suggesting that the kinase activity was limited by the amount of the cyclin B i n the cells. The growth of these cyclin B overexpressing mutants was arrested during mitosis. These mitotically arrested cells formed very small fruiting bodies when allowed to undergo development. A protein that eluted together with the histidine tagged cyclin B fusion protein from a Ni-resin affinity column was identified as Cdc2, indicating a direct physical interaction between cyclin B and Cdc2. Indirect immunofluorescence staining using the cyclin B antibody revealed that D. discoideum cyclin B localized mainly in the nucleus. This is consistent with the finding that D. discoideum cyclin B is lacking the presumptive cytoplasmic signal sequence found in most of the B-type cyclins from multicellular organisms.
Item Metadata
Title |
Isolation and functional studies of D. discoideum cyclin B gene during growth and development
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1995
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Description |
Cyclin B plays an essential role i n cell cycle regulation by complexing with
Cdc2 protein kinase to direct cells into mitosis. A cyclin B gene (cycB) has been
isolated from D. discoideum. The cyclin box region of the protein encoded by cycB has
a high degree of sequence identity with B-type cyclins of other species. During celldivision
synchronized growth, levels of cyclin B mRNA and protein were found to
oscillate during the cell cycle with maximum accumulation occurring prior to and
during cell division. Northern blot analysis showed that the cycB mRNA levels
peaked twice during development, once during early aggregation (3 h) and again at
the tipped aggregate stage (12 h). In contrast, the levels of cyclin B protein and
histone H I kinase activity of the Cdc2-immunoprecipitate increased only during early
development and then gradually decreased as development continued.
The N-terminal region of the cyclin B protein has the conserved protein
degradation motif, typical of cyclin B proteins. The 5' portion of the D. discoideum
cycB gene was deleted and the truncated gene was cloned downstream from the
regulatable discoidin promoter. Overexpression of this truncated cyclin B gene
elevated the histone H I kinase activity of the Cdc2-immunoprecipitates, suggesting
that the kinase activity was limited by the amount of the cyclin B i n the cells. The
growth of these cyclin B overexpressing mutants was arrested during mitosis. These
mitotically arrested cells formed very small fruiting bodies when allowed to undergo
development. A protein that eluted together with the histidine tagged cyclin B fusion protein
from a Ni-resin affinity column was identified as Cdc2, indicating a direct physical
interaction between cyclin B and Cdc2. Indirect immunofluorescence staining using
the cyclin B antibody revealed that D. discoideum cyclin B localized mainly in the
nucleus. This is consistent with the finding that D. discoideum cyclin B is lacking the
presumptive cytoplasmic signal sequence found in most of the B-type cyclins from
multicellular organisms.
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Extent |
12031017 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-02-19
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0087290
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1996-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.