A STUDY OF AN UNUSUAL CYTOSKELETON IN TWO SPECIES OF THE PHYTOFLAGELLATES OCHROMONAS (ACTIN, MICROFILAMENT, ELECTROPHORESIS).
Item
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Title
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A STUDY OF AN UNUSUAL CYTOSKELETON IN TWO SPECIES OF THE PHYTOFLAGELLATES OCHROMONAS (ACTIN, MICROFILAMENT, ELECTROPHORESIS).
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Identifier
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AAI8629665
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identifier
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8629665
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Creator
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ODDOUX, CAROLE.
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Contributor
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Sheldon Aaronson
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Date
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1986
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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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Chemistry, Biochemistry
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Abstract
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Relatively little work has been done on the cytoskeletal structure of lower eukaryotic or plant systems. While vertebrate systems may be easier to study because their tissue cells are specialized for a limited set of activities and cytoskeletal requirements, it is the lower eukaryotes that must engage in the total spectrum of activities, and therefore require multifunctional cytoskeletons. The complex and often unexpectedly different cytoskeletons of lower eukaryotes frequently makes these systems particularly difficult to study. Other complicating factors include the presence of active proteases, phenolases and pigments that may destroy proteins or interfere with standard biochemical techniques.;In this thesis, the cytoskeletons of two species of Ochromonas were studied. The adverse effects of proteolysis, phenolase activity and pigments on protein isolation and polyacrylamide gel electrophoresis were combatted by the use of the proteolytic inhibitors benzamidine, 1,10-orthophenanthroline and PMSF, and the use of the alkaline buffer system of Piccioni et al. (1982) "Methods in Chloroplast Molecular Biology", Elsevier Biomedical Press, N.Y. p 985. for electrophoresis.;Possible evidence for the presence of small amounts of actin was provided by positive anti-actin immunofluorescence. In addition, a protein comigrating with muscle actin eluted from a DEAE-cellulose column at a KCl concentration of 0.1 M - 0.35 M. A DNAase I inhibition activity also eluted in these fractions. Negative NBD-phallicidin results could indicate that the actin is present in non-filamentous form or in amounts below the detection limit of this assay. SEM and TEM of cold Triton X-100-extracted cells indicated the presence of a complex mesh-like cytoskeleton consisting of filaments of various sizes which are not accounted for by the tubulin or actin that could possibly be present. SDS-polyacrylamide gel electrophoresis revealed the presence of four major skeletal proteins of about 95.5 (or 111), 68, 40, and 30 kD. S1 labeling of Triton X-100-extracted cells was inconclusive because of the complex pattern created by the dense meshworks of these cytoskeletons.;Taken together, the results indicate that if actin is present, it is only present as a minor component of the cytoskeleton, possibly, in non-filamentous form. The complex cytoskeleton consists of four major proteins besides actin and tubulin. These proteins could possibly be intermediate filament-related or entirely new cytoskeletal proteins.
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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.
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Program
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Biochemistry
