Mapping surface proteins of Chlamydomonasreinhartdii vegetative, gametic and activated gametic cells.
Item
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Title
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Mapping surface proteins of Chlamydomonasreinhartdii vegetative, gametic and activated gametic cells.
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Identifier
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AAI9720149
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identifier
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9720149
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Creator
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Viskaduraki, Maria Fotios.
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Contributor
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Adviser: Charlene Forest
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Date
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1997
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Cell | Biology, Microbiology
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Abstract
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Biotinylation of surface proteins, plasma membrane isolation and two-dimensional gel electrophoresis have been employed to map Chlamydomonas plasma membrane proteins. Vegetative cells, unactivated and db-cAMP activated gametes of both mating types were labeled with sulfo-NHS-biotin, washed, deflagellated and disrupted. The plasma membranes were purified and after solubilization the plasma membrane proteins were subjected to 2-D gel electrophoresis and western transfer. Biotinylated proteins were detected with streptavidin-alkaline phosphatase. Comparison of vegetative, unactivated and activated gametic maps gave a clear picture of the changes occurring during gametogenesis and mating structure activation and stage and mating type specific proteins were identified.;In comparisons of {dollar}mt\sp+{dollar} and {dollar}mt\sp-{dollar}, vegetative cells of the two mating types differ in greater than 50% of their polypeptides. If gametes of the two mating types are compared, less than 1/3 of the biotinylated polypeptides are shared between {dollar}mt\sp+{dollar} and {dollar}mt\sp-{dollar}. Whether these differences between mating types are due to mating type or strain variation is under investigation in our lab.;Analysis of the developmental process in each mating type shows that vegetative and gametic plasma membranes have very different polypeptide patterns. While 50% of the {dollar}mt\sp+{dollar} vegetative spots are lost during gametogenesis, for {dollar}mt\sp-{dollar} there is a 60% loss of vegetative polypeptides. Approximately 50% of the gametic polypeptides are newly appearing ones in both mating types. This suggests that there is a large turnover of plasma membrane polypeptides during gametogenesis.;When gametes of individual mating types are activated with db-cAMP, a few new polypeptides are detected. 61% of the activated {dollar}mt\sp+{dollar} and 77% of the activated {dollar}mt\sp-{dollar} polypeptides come from unactivated gametes. Of the newly detected polypeptides of activated gametes, 7 were found to be specific to {dollar}mt\sp+{dollar} and 6 were found to be specific to {dollar}mt\sp-{dollar}. These polypeptides specific for the activated gametes of one mating type may include polypeptides that are involved in the mating process.;This system will be useful for studying the roles of plasma membrane proteins in mating of this organism. Mutants defective in a number of stages in the mating reaction are available and can be used for this purpose.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
