Doctoral Dissertations

Date of Award

8-1985

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Donald E. Olins

Committee Members

Robert Fujimura, Ed Grell, Ada L. Olins

Abstract

Macronuclear replication bands (RB) of the hypotrichous ciliated protozoa were studied by cytochemical, biochemical and immunochemical methods. The cytochemistry of the RB was examined using the thiol specific fluorochrome coumarin maleimide and a modification of the silver staining techniques for nucleolar organizing regions (NORs). An abundance of thiol groups and silver staining proteins were demonstrated in the RBs and nucleoli of Euplotes eurystomus, Stylonychia mytilus and Oxytricha nova. The silver staining proteins in the RB were differentially extracted with acid, without any decrease of nucleolar staining. Triton-acid-urea gel electrophoresis of an acid extract of macronuclei revealed enhanced silver reaction with a single protein upon selective silver staining. The thiol proteins were not acid extractable and the staining reaction depended on the availability of free reactive sulfhydryl groups. In order to study the biochemistry of the RB, a method was developed for isolating RBs from Euplotes. The isolated RBs retained the cytochemical properties of staining with silver and coumarin maleimide. A method was also described for isolating highly purified macronuclei. Ultrastructural examination of RBs and macronuclei demonstrated that the morphology remained essentially intact during purification. Replicating molecules were enriched in RBs compared to macronuclei. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis also demonstrated an enrichment of a 55 kilodalton protein in RBs when compared to macronuclear proteins. Both isolated RBs and macronuclei were composed of gene-sized chromatin. The RBs of Euplotes were further characterized using monoclonal antibodies (MAbs) prepared from mice immunized with isolated RBs. A panel of eight MAbs were selected by radioimmunoassay (RIA) using soluble chromatin from isolated RBs or macronuclei as antigen. Several MAbs recognized antigens present only in the RB or macronucleus and others recognized antigens present in both structures. Four MAbs gave positive immunofluorescent staining. Antibody ClO recognized an antigen in the rear zone of the RB, while MAbs G6 and B2 stained both zones. Antibody A7 reacted with an epitope distributed throughout the macronucleus except for the RB. All four MAbs reacted with antigens composed of peptides. The proteins reacting with some of the RB specific MAbs were identified on immunoblots.

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