Degree Type


Date of Award


Degree Name

Doctor of Philosophy




An enrichment selection method was used to isolate Chinese hamster ovary (CHO) AK 412 variants resistant to 1 (mu)g/ml cytochalasin D (CD). The cytochalasin D-resistant (Cyd('R)) variant phenotype was thoroughly characterized by physiological, cytogenetic, morphological and biochemical techniques.;The Cyd('R)-variants were shown to possess a membrane lesion which confers CD resistance by reducing the uptake of CD from the extracellular medium. The change in membrane structure conferring CD resistance was correlated with several changes in membrane function, including: a generalized drug cross-resistance; hypersensitivity to a membrane specific drug (procaine); significant changes in cell surface morphology; differences in cellular adhesion properties; and a decrease in membrane pliability.;Cell surface proteins (glycoproteins) were radiolabeled by three different methods: (1) ('125)I-lactoperoxidase catalyzed iodination; (2) labeling of surface galactose residues by ('3)H-NaBH(,4)-galactose oxidase; and (3) metabolic labeling of cell surface glycoproteins with ('3)H-fucose and ('3)H-glucosamine. Autoradiograms from SDS-polyacrylamide gels of labeled membrane proteins revealed at least six differences between the Cyd('R)-variants and the CHO parent. In all cases, the labeled proteins migrated slightly slower in the variant, suggesting that increased glycosylation and/or some other modification of membrane glycoproteins is associated with CD-resistance.;Although no definitive genetic mechanism or gene product responsible for CD resistance was established, indirect evidence strongly suggests that CD resistance (and other characteristics associated with the Cyd('R)-phenotype) is mediated by gene amplification. The amplified gene exists in the form of a novel homogeneous staining region (HSR) on the long arm of chromosome 1.;The characteristics associated with the Cyd('R)-phenotype have been observed in many other drug-uptake and efflux mutants. A model is proposed that attempts to explain reduced drug uptake or increased drug efflux in terms of gene amplification. In this model, amplification of a gene(s) that controls glycosylation and/or phosphorylation levels of cell surface glycoproteins results in a pleiotropic phenotype.



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Stephen Howard Grund



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277 pages

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Genetics Commons