Evidence for an actomyosin system in Tetrahymena.
Item
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Title
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Evidence for an actomyosin system in Tetrahymena.
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Identifier
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AAI9405534
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identifier
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9405534
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Creator
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Hoey, John Gabriel.
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Contributor
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Adviser: Ray H. Gavin
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Date
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1993
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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, Cell | Biology, Molecular
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Abstract
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Affinity-purified antibodies were used in conjunction with immunofluorescence and immunogold microscopy to study the distribution of actin in Tetrahymena and to provide evidence for a putative actomyosin system in this organism. An antiserum against a Tetrahymena oral apparatus fraction enriched for basal body proteins was produced in rabbits. Agarose-linked muscle actin was used to affinity-purify anti-Tetrahymena actin antibodies from the oral apparatus antiserum. The affinity-purified antibodies were monospecific for Tetrahymena actin on immunoblots containing total oral apparatus protein. Agarose-linked myosin II light chains were used to affinity-purify antibodies that detected an 18 kDa Tetrahymena polypeptide on immunoblots but did not cross react with actin. With immunofluorescence microscopy, the anti-actin antibodies localized to basal bodies in Tetrahymena. Tetrahymena basal bodies are contained within separate, filamentous cages which are closely associated with basal body microtubules. At the ultrastructural level with the immunogold labeling technique, the affinity-purified, anti-actin antibodies labeled actin epitopes in four distinct regions of the basal body-cage complex: (a) basal body walls, (b) basal plate filaments, (c) proximal-end filaments, and (d) cage wall filaments. The anti-actin antibodies also localized to mucocysts (cortical secretory organelles), and to the fibrillar macronuclear matrix. Quantitative analysis of gold particle distribution was used to demonstrate the specificity of the antibodies for the basal body-cage complex, mucocysts, and macronuclear matrix and to show that non-specific binding of the antibodies was negligible. Preadsorption of the antibody with muscle actin effectively eliminated the capacity of the antibody to bind to proteins on immunoblots and to basal body structures with the immunogold labeling technique. The anti-18 kDa antibody and the anti-actin antibody co-localized to components of the basal body-cage complex. A Tetrahymena cortical fraction containing basal bodies exhibited both Mg{dollar}\sp{lcub}2+{rcub}{dollar}-EGTA and K{dollar}\sp+{dollar}-EDTA ATPase activity. Approximately 60% of the Mg{dollar}\sp{lcub}2+{rcub}{dollar}-EGTA ATPase activity was vanadate-insensitive. The data in this study provide evidence for an actomyosin system in Tetrahymena which could play a role in basal body movement and in the discharge of mucocyst contents.
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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.
